Biologically active taxane analogs and methods of treatment by oral administration

ABSTRACT

The present invention relates to a novel chemical compound for use in the treatment of cancer, to compositions containing said compound, methods of manufacture and combinations with other therapeutic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/950,316 filed Jul. 25, 2013, which is a continuation of U.S. patentapplication Ser. No. 13/620,852 filed Sep. 15, 2012 now granted as U.S.Pat. No. 8,552,056, which is a continuation of U.S. patent applicationSer. No. 12/873,477 filed Sep. 1, 2010 now granted as U.S. Pat. No.8,273,789, which is a continuation of U.S. patent application Ser. No.12/593,153 which is now abandoned, which is 371 U.S. National Stage ofPCT/US2008/055384 filed Feb. 28, 2008 now expired, which claims priorityto and claims the benefit of U.S. Provisional Application No. 60/908,459filed Mar. 28, 2007 now expired and U.S. Provisional Application No.60/911,607 filed Apr. 13, 2007 now expired, all of which areincorporated herein by reference in their entireties.

The present invention relates to a novel chemical compound for use inthe treatment of cancer, to compositions containing said compound,methods of manufacture and combinations with other therapeutic agents.

Various taxane compounds are known to exhibit anti-tumor activity. As aresult of this activity, taxanes have received increasing attention inthe scientific and medical community, and are considered to be anexceptionally promising family of cancer chemotherapeutic agents. Forexample, various taxanes such as paclitaxel and docetaxel have exhibitedpromising activity against several different varieties of tumors, andfurther investigations indicate that such taxanes promise a broad rangeof potent anti-leukemic and tumor-inhibiting activity. One approach indeveloping new anti-cancer drugs is the identification of superioranalogs and derivatives of biologically active compounds. Modificationsof various portions of a complex molecule may lead to new and betterdrugs having improved properties such as increased biological activity,effectiveness against cancer cells that have developed multi-drugresistance (MDR), fewer or less serious side effects, improvedsolubility characteristics, better therapeutic profile and the like.

International Patent Application WO 2005/030150 discloses a series oftaxane analogues useful for the treatment of cancer as well as methodsof producing them. Other taxanes are described in EP 1 228 759; EP 1 285920; EP 1 148 055; WO 01/56564; WO 01/57027; WO 94/10996; FR 2 715 846;U.S. Pat. No. 5,352,806; FR 2 707 293; WO 94/08984; WO 92/09589; WO94/20485; WO 93/21173; Klein L L, “Synthesis of 9-Dihydrotaxol: a novelbioactive taxane”, Tetrahedron letters, vol. 34, no. 13, 1993, pages2047-2050; Datta A et al, “Synthesis of novel C-9 and C-10 modifiedbioactive taxanes”, Tetrahedron letters, vol. 36, no. 12, 1995, pages1985-1988; Klein L L et al, Journal of Medicinal Chemistry, no. 38,1995, pages 1482-1492; J Demattei et al, “An efficient synthesis of thetaxane-derived anticancer agent abt-271”, Journal of Organic Chemistry,vol. 66, no. 10, 2001, pages 3330-3337; Gunda I Georg et al, “Thechemistry of the taxane diterpene: stereoselective reductions oftaxanes”, Journal of Organic Chemistry, vol. 63, no. 24, 1998, pages8926-8934. However, apart from paclitaxel and dodetaxol, no taxane hasreceived regulatory approval to be marketed for use as an anticanceragent and no taxane has received regulatory approval to be marketed foruse by oral administration. WO 2005/03150 and US Application No2005/0148657 A1 disclose taxane analogues and derivatives possessing a9a, 10a configuration and an acetal or ketal bridge between the hydroxylgroups at the 7- and 9-positions. Synthesis of taxane analogues is alsodisclosed in WO 2007/073383, WO 2007/126893 and WO 2007/075870.

Zamir et al, Tetrahedron Letters, 37, 6435-6438 (1996) discloses taxaneanalogues containing a five membered A-ring and a position 1 C(CH₃)₂OHgroup. These analogues were abeo-taxanes lacking the four memberedoxatane ring. The compounds possessed a 10β-, stereochemistry. Noanti-cancer activity was ascribed to the compounds. Zamir et al,Tetrahedron Letters, 53. 15991-16008 (1997) discloses abeo-taxaneanalogues and also trapped intermediates containing a 5 membered A-ringand a position 1 C(CH₃)₂OH group. The compounds possessed a10β-stereochemistry and an acetoxy group at position 13. Numerousstructural analogues were disclosed but no biological data was providedon any compound. Wahl et al, Tetrahedron, 48, 6965-6974 (1992) disclosesa wide range of modified taxane analogues including one which had a 5membered A-ring and a 1 position C(CH₃)₂OH group. The compound has a10β-hydroxyl group and a 9-keto group and was said to be active in thetubulin disassembly assay although no data was provided. Otherpublications relating to taxane analogues include Hue et al, Magn. ResonChemis, 45, 527-530 (2007); Nicolaou et al, Mg. Chem. Int. Ed. 44,1012-1044 (2005); Zamir et al, Tetrahedron Letters, 40, 7917-9920(1999); Torregiani et al, Gaz. Chim. Italiana, 126, 809-814 (1996);Appendino et al, J. Chem. Soc. Comm., 1587-1589 (1993); and Samaranayakeet al, J. Org. Chem., 56, 5114-5119 (1991). There is a need in the fieldof anti-cancer therapy for new therapeutic agents having improvedpharmacological properties, such as increased biological activity(efficacy), effectiveness against cancer cells having multi-drugresistance (MDR), fewer or less serious side effects, improvedsolubility characteristics, better therapeutic profile and the like. Ofparticular need is the development of drugs having improved MDR reversalproperties. There is also a need for methods of treating patients withsuch compounds in cancer treatment regimens. Many anti-cancer drugscurrently available have utility against only a limited number of cancercell-lines and there is therefore a need for new treatments that haveutility against a wider range of cancers and specifically against thosecancers which are currently difficult to treat or for which noappropriate therapy is available.

It is also the case that the majority of anti-cancer drugs must beadministered by infusion or injection, and there exists a need for drugsthat can be more readily administered to patients, in particular,orally. Furthermore, an ideal drug candidate will exist in a physicalform that is stable, non-hygroscopic and easily formulated. The presentinvention is directed to meeting these needs, among other needs asdisclosed herein.

Accordingly, the present invention provides a compound as a singlediastereoisomer of the formula S-(1):

In another aspect the present invention also provides a pharmaceuticalcomposition comprising a compound of formula S-(1) as herein describedtogether with a pharmaceutically acceptable diluent or carrier. Thecompound and compositions of the present invention have utility in thetreatment of cancer. In another aspect of the invention therefore isprovided the compound and compositions of the invention for use in thetreatment of cancer. The present invention provides a method of treatingcancer in a patient comprising administering to the patient acomposition comprising the compound of the invention as hereinbeforedescribed.

As used herein, the terms “taxane,” “taxanes,” “taxane derivatives” or“taxane analogs” and the like, include the diterpenes produced by theplants of the genus Taxus (yews), and may be derived from naturalsources, may be prepared synthetically or may be obtained fromsemi-synthetic methods or a combination thereof. Such taxanes includepaclitaxel and docetaxel that have a 6-membered A ring, as well as theabeo-taxanes that have a 5-membered A ring, as known in the art and asdisclosed herein. The acid catalyzed rearrangement from the 6 membered Aring (and 8 membered B ring) to the 5 membered A ring (and 7 membered Bring) of the abeo taxane has been described for other taxane compounds,for example, in L. O. Zamir et al, Tetrahedron Letters, 40 (1999)7917-7920, L. O. Zamir et al, Tetrahedron, Vol. 53, No. 47, 15991-16008(1997), L. O. Zamir et al, Vol. 37, No. 36, 6435-6438 (1996), A. Wahl etal, Tetrahedron, Vol. 48, No. 34, 6965-6974 (1992), G. Appendino et al,J. Chem. Soc., Chem. Commun. 1587-1589 (1993), and G. Samaranayake etal, J. Org. Chem. 1991, 56, 5114-5119. The nomenclature used in thesepublications to name the rearranged 5 membered A ring structures hasbeen 11(15->1) abeo-taxanes.

The term “baccatin” or “baccatin derivatives” means the taxanederivatives in which the side chain at the 13-position of the taxaneskeleton is a hydroxy group and these derivatives are often referred toin the literature as a baccatin or “baccatin I-VII” or the likedepending, on the nature of the substituents on the tricyclic rings ofthe taxane skeleton.

The term “subject” as used herein refers to an animal, preferably amammal, most preferably a human.

The term “brain cancer” or “brain tumor” refers to any tumor that growsin the brain, including, but not limited to, astrocytoma,craniopharyngioma, glioma, ependymoma, neuroglioma, oligodendroglioma,glioblastoma multiforme, meningioma, medulloblastoma and other primitiveneuroectoderma.

“Therapeutically effective amount” or an “effective amount” means a drugor composition of the present application that elicits any of thebiological effects as listed in the present specification.

Brain disease further refers to cancers which have metastasized to thebrain.

Pharmaceutically acceptable excipient, diluent, carrier or salt as usedherein, means the excipient, diluent, carrier or salt of the compoundsdisclosed herein, that are pharmaceutically acceptable and provides thedesired pharmacological activity.

Pharmaceutically acceptable salts of the compound of formula S-(1)comprise base salts thereof. Suitable base salts are formed from baseswhich form non-toxic salts. Examples include the aluminum, arginine,benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine,magnesium, meglumine, olamine, potassium, sodium, tromethamine and zincsalts. Hemisalts of bases may also be formed, for example, hemisulphateand hemicalcium salts. For a review on suitable salts, see Handbook ofPharmaceutical Salts: Properties, Selection, and Use by Stahl andWermuth (Wiley-VCH, Weinheim, Germany, 2002).

Purity of diastereoisomers relates to the amount of a particulardiastereoisomer that is present with respect to other diastereoisomers.

International Patent Application Nos. WO 2005/030150 and WO 2007/126893and US Patent Application Nos. US 2005/148657 and US 2007/225510disclose processes which lead to the preparation of the compound offormula (I), although the structural formula was not provided.

This compound consists of a mixture of compounds of formula S-(1) andR-(1):

In attempting to separate these diastereoisomers, it was discovered thatstandard scale up chromatographic methods for the purification andseparation of isomers using silica gel of various grades, from 28-200mesh, 100-200 mesh, and Davisil® grade 633, 200-425 mesh, C-18 reversedphase media and attempted crystallization with various solventscompositions and solvent mixtures did not provide efficient separationof the diastereomeric mixture. Solvents such as hexanes, ethyl acetate,methyl tert-butyl ether, ethanol, acetone and their mixtures indifferent ratios and compositions were determined to be ineffective forthe separation of each of the diastereoisomers from each other. It wassurprisingly discovered that a solvent composition of 40% MTBE inheptane gave indication of separation of the diastereoisomers on asilica HPTLC plate with concentration zone with the compound of formulaS-(1) showing an Rf ˜0.15. Based upon this observation, it was electedto attempt a normal phase chromatographic separation of thediastereoisomers by normal phase column chromatography. Having succeededin separating the diastereoisomers it was suprisingly discovered thatthe compound of formula R-(1) had better solubility properties than thecompound of formula S-(1) which is an important beneficial property intaxane cancer drugs.

It was surprisingly discovered that the compound of formula S-(1)possessed biological properties that were not obvious in light of thoseof the mixture of diastereoisomers identified as formula (1). Afterhaving separated and identified the individual diastereoisomers it wassurprisingly discovered that the S-(1) diastereoisomer was significantlymore active than the diastereomeric mixture. It is therefore preferableto use this diastereoisomer even though the R-(1) diastereoisomer hasbetter solubility properties.

The pancreas can be divided into two parts: the exocrine and endocrinepancreas. About 95% of pancreatic cancers begin in the exocrinepancreas. The rest are cancers of the endocrine pancreas, which are alsocalled islet cell cancers. Accordingly the compound and compositions ofthe present invention have utility in the treatment, preferably oraltreatment, of these cancers.

In the brain, the design and development of effective anti-tumor agentsfor treatment of patients with malignant neoplasms of the centralnervous system have been influenced by two major factors: 1) the drugsgiven at high systemic levels are generally cytotoxic; and 2) theblood-brain barrier provides an anatomic obstruction, limiting access ofdrugs to these tumors. Accordingly, one problem posed by the blood-brainbarrier is that, in the process of protecting the brain, is that itexcludes many potentially useful therapeutic agents.

The chemotoxic effect of taxanes, such as paclitaxel, is a result of theability to inhibit cellular mitosis. Paclitaxel acts by binding to theβ-subunit of polymerized tubulin, promoting the assembly of microtubulesand inhibiting the normal dynamics of tubulin assembly necessary formitosis. Resistance to taxanes is associated with the expression of theMDR 1 gene encoding P-gp, an ATP-dependent drug efflux pump thattransports paclitaxel, other taxanes and other cytotoxic agents out ofcells. The compound of formula S-(1) according to the present inventionis a novel taxane analog, which achieves mutant-β-tubulin binding. Inaddition the compound of the invention, unlike paclitaxel, is not asubstrate for the MDR protein. Modulation of the P-glycoprotein, whosesubstrates are actively pumped out of brain cells into capillary lumens,has been found to facilitate the delivery of drugs to the brain. Asdemonstrated by the experiments described herein, the compound offormula S-(1) freely crosses the blood-brain barrier and has activityagainst brain tumors.

Surprisingly, in xenograft models, the compound of formula S-(1) hasbeen shown to be active as an antitumor agent against xenografts,regardless of whether such xenografts express the P-gp pump. In additionto this anti-resistance activity, the compound of formula S-(1) alsoappears to have a number of advantages over traditional taxanes,including activity in brain tumors, pancreatic tumors, and oralbioavailability.

Furthermore, as shown in Table 1, the compound of formula S-(1) wasdetermined to be significantly more active than the diastereoisomer offormula R-(1) in a number of cytotoxicity assays as measured by MTSproliferation assay.

TABLE 1 MTS Proliferation Assay Results nM IC₅₀ Cancer Type & Cell lineS-(1) R-(1) Neuroblastoma 11 80 SKNAS head/neck 21 96 FADU prostate 60212 DU145 breast 10 52 MDA435s head/neck 9.4 37.7 KB head/neck 553 1196KBV (MDR+) colon 0.006 1.6 HT29 uterine 2.9 7.8 MESSA uterine 44.8 233MESSA/Dox (MDR+) prostate 0.03 2.5 PC-3

In one embodiment of the present invention there is provided thecompound of formula S-(1) as a single diasteroisomer as shown below.Also provided herein are novel methods for the preparation of S-(1),pharmaceutical compositions comprising S-(1) and methods of treatingcancer comprising administering such a pharmaceutical composition to apatient in need thereof.

In a preferred embodiment the compound is isolated as a purediastereoisomer. Preferably the isolated compound is greater than 95%pure. More preferably the compound is greater than 99% pure. Preferably,therefore, the present invention encompasses the compound of formulaS-(1) as a single diastereoisomer having a diastereomeric excess (d.e.)of at least 80%, preferably at least 90%, more preferably at least 95%,yet more preferably at least 98% and most preferably at least 99%. Inparticular, the compound of the invention comprises less than 5% of thediastereoisomer of formula R-(1), preferably less than 3%, morepreferably less than 2%, yet more preferably less than 1% and mostpreferably less than 0.5%.

Additionally, the compound of the present invention comprises less than5% of the compound of formula (2), preferably less than 3%, morepreferably less than 2%, yet more preferably less than 1% and mostpreferably less than 0.5%.

In another aspect of the invention there is provided a pharmaceuticalcomposition comprising the compound of formula S-(1) as herein definedin combination with a pharmaceutically acceptable diluent or carrier.Additionally, the compound of formula S-(1) as herein defined may beadministered orally. The invention therefore also provides apharmaceutical composition for oral administration comprising thecompound of formula S-(1) as herein defined in combination with apharmaceutically acceptable diluent or carrier. Additionally, thecompound of formula S-(1) as herein defined may be administeredparenterally, such as by intravenous injection or infusion. Theinvention therefore also provides a pharmaceutical composition forparenteral administration comprising the compound of formula S-(1) asherein defined in combination with a pharmaceutically acceptable diluentor carrier.

In another aspect there is provided a method for the treatment of cancerin a patient comprising administering to the patient a therapeuticallyeffective amount of the compound of formula S-(1) as herein defined to apatient in need of such treatment. The compound of the present inventionmay be used in the treatment of diseases mediated by tubulin. The cancermay be selected from the group consisting of brain, hepatocellular,sarcoma, leukemia, lymphoma, and other bone marrow dyscrasias,neuroblastoma, glioblastoma, cervical, colorectal, pancreatic, renal,thyroid, lung, small-cell lung, non-small cell lung, gastric, breast,ovarian, prostate, head and neck and melanoma. In one preferredembodiment the cancer is colorectal cancer. In another preferredembodiment the cancer is pancreatic cancer. In another preferredembodiment the cancer is prostate cancer. In yet another preferredembodiment the cancer is brain cancer. In a most preferred embodimentthe cancer is neuroblastoma or glioblastoma.

The present application provides pharmaceutical compositions comprisinga diasteroisomer of the formula S-(1) in which the isomer is greaterthan 90% pure, greater than 95% pure, greater than 97% pure, greaterthan 99% pure or greater than 99.5% pure. The present inventionadditionally comprises compounds useful as intermediates in processesfor the preparation of the compound of formula S-(1).

In one embodiment of the present invention there is provided thecompound of formula S-(34) as a single diasteroisomer as shown below.Also provided herein are novel methods for the preparation of S-(34).

Preferably, the present invention encompasses the compound of formulaS-(34) as a single diastereoisomer having a diastereomeric excess (d.e.)of at least 80%, preferably at least 90%, more preferably at least 95%,yet more preferably at least 98% and most preferably at least 99%.

In particular, the compound of the invention comprises less than 5% ofthe diastereoisomer of formula R-(34), preferably less than 3%, morepreferably less than 2%, yet more preferably less than 1% and mostpreferably less than 0.5%.

Processes

Methods for the preparation of the compound of formula (1) are describedin International Patent Applications WO 2005/030150 and WO 2007/126893.Although the structure was not provided these processes lead to itspreparation.

Definitions

CSA: Camphorsulfonic Acid

DCC: N,N′-Dicyclohexyl-Carbodiimide

10-DAB III: 10-Deacetylbaccatin III

DCM: Dichloromethane

DMAP: Dimethylaminopyridine

DMSO: Dimethylsulfoxide

Hr: Hour

HPLC: High Performance Liquid Chromatography

IPAc: Isopropyl Acetate

LCMS: Liquid Chromatography Mass Spectrometry

MTBE: Methyl tert-Butyl Ether

4-PP: 4-Pyrrolidinopyridine

TEA: Triethylamine

TES-Cl: Triethylsilyl Chloride

THF: Tetrahydrofuran

TPAP: Tetrapropyl Ammonium Perruthenate

TPAP: Tetrapropyl Ammonium Perruthenate

A method for synthesizing the compound of formula (3) is shown belowwith reference to Scheme 1. 10-Deacetylbaccatin III (10-DAB III), whichhas formula (4) as shown in Scheme 1, is a commercially available(Sigma-Aldrich) compound used as an intermediate in the preparation ofvarious taxanes.

In this process, 10-DAB III, formula (4), is first protected at both theC-7 and C-10 positions to form the C7, C10 di-CBZ derivative of formula(5). 10-Deacetylbaccatin III of formula (4) (50 g, 91 mmol) wasdissolved in THF (2 L, 40 ml/g) by warming to 40° C. in a warm-waterbath. The solution was cooled to −41° C. in a Neslab chiller andbenzylchloroformate (46 mL, 3.2 eq, 294 mmol) was added to the stirredchilled solution followed by further cooling to −44° C. To this solution2.3M hexyl lithium solution (130 mL, 3.3 eq, 303 mmol) was addedgradually over 45 min while maintaining the temperature of the reactionmixture at ≤−39° C. Stirring was continued in the Neslab for 45 minutesat which time HPLC indicated the reaction had gone to completion. At twohours total reaction time, the reaction was quenched by the addition ofIN HCl (400 mL) and IPAc (1 L) and removal from the Neslab chiller. Thereaction was allowed to stir while warming to 10° C. The layers wereseparated and the IPAc layer was washed sequentially with H₂O (500 mL),saturated NaHCO₃ (200 mL) and H₂O (4×500 mL) and then filtered through asilica gel pad. The filtrate was concentrated until solids started toform. IPAc (850 mL) was added and the mixture was heated to 60° C. todissolve some of the solids. To the warm solution, heptanes (800 mL)were added and the solution was cooled in the refrigerator and filtered.The solids collected by the filtration were washed with heptanes anddried under vacuum at 45° C. to give formula (5).

Next, the compound of formula (5) was coupled with a side chain to formthe compound of formula (7). Here, the side chain of the compound offormula (6), (38 g, 99.6 mmol) was dissolved in toluene to a knownconcentration (0.0952 g/mL). This solution was added to the compound offormula (5) (54.0 g, 66.4 mmol). The solution was heated in a warm-waterbath and DMAP (8.13 g, 66.4 mmol) and DCC (25.3 g, 120 mmol) in toluene(540 mL) were added to the warm reaction mixture. While maintaining thetemperature at about 51° C., the reaction was continually stirred andsampled periodically for HPLC. After 3 hours, additional DCC (13.0 g) intoluene (140 mL) was added. The following morning (25.25 hr), MTBE (450mL) was added and the reaction mixture was filtered through a pad ofsilica gel, washed with MTBE followed by ethyl acetate, and concentratedto give the compound of formula (7) as 61.8 g of an oil.

The compound of formula (7) was then deprotected at both the C7 and C10positions to give the compound of formula (8). A solution of THF (300mL) and HCl (22 mL) was added to a solution of the compound of formula(7) (61.8, 52.5 mmol) in THF (15 mL/g, 920 mL). The resulting solutionwas flushed with nitrogen. A catalyst (10% Pd/C with 50% water, 99.1 g)was added and the flask was flushed with nitrogen three times and thenwith hydrogen three times. The reaction mixture was stirred vigorouslyunder a hydrogen balloon for 21 hours. At this time the reaction wassampled and HPLC indicated that 38% by area of starting material stillremained. Water (10 mL) was added and stirring continued. Twenty hourslater, HPLC indicated the same amount of starting material stillremaining. The reaction mixture was filtered through celite and washedwith THF. It was then concentrated to remove excess THF; fresh catalyst(101 g) was added and the reaction mixture was placed back underhydrogen as before. After another 24 hours, an intermediate compound wasstill present and still more catalyst (20 g) was added. After anotherhour, HPLC indicated that the reaction was complete. The reactionmixture was filtered through celite and washed through with IPAc. Thecombined filtrate was washed with NH₄Cl solution (500 mL), water (500mL), 5% NaHCO₃ (500 mL), H₂O (300 mL), and brine (300 mL). The organiclayer was dried, filtered, and concentrated to give a foam of thecompound of formula 38 (42.5 g).

The compound of formula (8) was then converted to the compound offormula (9). Formula (8) (41.4 g, 52.5 mmol) was dissolved in DCM (500mL) at room temperature. In the case that the impurity was water, Na₂SO₄was added to the solution, and the solution was filtered through filterpaper into to a 2 L flask. The solids were collected and washed with DCM(250 mL) and the washings transferred into the flask. The flask wascovered with a septum and N₂ balloon. TEA (35 mL) followed by DMAP (1.28g) and TES-Cl (˜30 mL, 3.5 eq) were added to the solution and stirred.Additional TES-Cl (15 mL) and TEA (20 mL) were added, and after 6 hoursHPLC indicated the reaction had gone to completion.

The reaction was then quenched by the addition of ethanol (25 mL). Thelayers were separated and the organic layer was washed with saturatedNH₄Cl (˜500 mL). The organic layer was dried over Na₂SO₄ andconcentrated. A flash column was packed with silica gel and wet with 8:2heptane/IPAc (1.5 L). The solids were dissolved in 8:2 heptane/IPAc (250mL) and filtered to remove solids that would not dissolve. This solutionwas concentrated to ˜100 mL and applied to the column. The column waseluted with 8:2 heptane/IPAc and fractions collected. Fractions withproduct were pooled and concentrated to give foam of formula (9) (24.5g).

The compound of formula (9) was then oxidized to form the compound offormula (10). Here, solid Na₂SO₄ was added to a solution of formula (9)(24.5 g, 24.0 mmol) and 4-methyl morpholine N-oxide (10.1 g, 84 mmol) inDCM (340 mL) to assure that the reaction was dry. The mixture wasstirred for 1 hour and then filtered through 24 cm fluted filter paperinto a 2 L 3-neck round bottom flask. The Na₂SO₄ solids were washed withDCM (100 mL) and the washings transferred into the flask. Molecularsieves (6.1 g, 0.15 g/g) were added to the solution and stirring wasbegun. TPAP (1.38 g) was added and the reaction was allowed to stirunder a N₂ blanket. Samples were taken periodically for HPLC. AdditionalTPAP (0.62 g) was added after 2 hours and again (0.8 g) after 15 hours.The reaction mixture was applied to a pad of silica gel (86 g), wet with8:2 heptane/IPAc and eluted with IPAc. The fractions were collected,pooled and concentrated to an oil. 4-Methyl morpholine N-oxide (5.0 g)and DCM (100 mL) were added and stirred. Na₂SO₄ (13 g) was added to themixture and it was filtered through filter paper. The Na₂SO₄ solidsremaining in the filter was washed with DCM (45 mL). Molecular sieves (5g) and TPAP (1.03 g) were added to the solution and after 45 minutes,more TPAP (1.05 g) was added. A pad of silica gel was prepared and wetwith 80:20 Heptane/IPAc. The reaction mixture was applied to the pad andeluted with IPAc. Fractions were collected and those fractionscontaining product were pooled and concentrated to give an oil productof formula (10) (21.8 g). Next, the compound of formula (10) was reducedto form the compound of formula (11). NaBH₄ (365 mg, 6 eq) was added toa stirred solution of formula (10) (1.6 g) in ethanol (19 mL) andmethanol (6.5 mL) cooled in an ice-water bath. After 1 hour, thereaction mixture was removed from the ice-water bath and at 2 hours, thereaction was sampled for HPLC, which indicated the reaction had gone tocompletion. The reaction mixture was cooled in an ice-water bath and asolution of NH₄OAc in methanol (15 mL) was added followed by theaddition of IPAc (50 mL) and H₂O (20 mL). It was mixed and separated.The organic layer was washed with water (20 mL) and brine (10 mL), asecond time with water (15 mL) and brine (10 mL), and then twice withwater (2×15 mL). It was dried over Na₂SO₄ and placed in the freezerovernight. The following morning a sample was taken for HPLC and thereaction was dried and the organic layer was concentrated on the rotaryevaporator. It was placed in the vacuum oven to give a foam product offormula (11) (1.45 g).

The compound of formula (11) was then acylated to form the compound offormula (12). TEA (5.8 mL, 41.5 mmol), Ac₂O (2.62 mL, 27.7 mmol) andDMAP (724 mg, 5.5 mmol) were added to a solution of formula (11) (14.1g, 13.8 mmol)) in DCM (50 mL). The reaction was stirred and sampled forHPLC periodically. After 18.5 hours, additional TEA (1.5 mL) and Ac₂O (1mL) were added. At 19 hours, HPLC indicated the reaction had gone tocompletion. The reaction mixture was diluted with IPAc (300 mL) andpoured into 5% NaHCO₃ (100 ml). It was then stirred, separated, and theorganic layer was washed with water (100 mL), saturated NH₄Cl (2×100mL), water (3×50 mL) and brine (50 mL) and then filtered through Na₂SO₄.The mixture was concentrated to give a foam product of formula (12)(14.6 g).

Next, the compound of formula (12) was converted to the compound offormula (13). A quantity of formula (12) (3.0 g, 2.83 mmol) was weighedinto a 100 mL flask. Next, DCM (24 mL) followed by methanol (6 mL) wereadded to the flask at room temperature. Stirring of the mixture beganunder N₂ and camphorsulfonic acid (CSA) (0.0394 g, 0.17 mmol) was added.After 4 hours LCMS indicated the product had formed. 5% NaHCO₃ (15 mL)was added to the reaction mixture; it was shaken vigorously and thentransferred to a separatory funnel. The reaction flask was rinsed intothe separatory funnel with 5% NaHCO₃ (25 mL) and, thereafter, thereaction mixture was shaken and the layers were separated. The organiclayer was washed with brine, dried over Na₂SO₄, and concentrated. MTBE(3×25 mL) was added and the reaction mixture was concentrated to drynessafter each addition to finally give 3.71 g foam. The foam was dissolvedin MTBE (10 mL) and stirred. Heptane (50 mL) was slowly added to thereaction solution and solids began to form immediately. The solids werevacuum filtered and rinsed with heptane (720 mL). The solids werecollected and dried in a vacuum oven at 40° C. to give the compound offormula (13) (2.18 g).

The compound of formula (13) was then converted to the compound offormula (3). A solution of formula (13) (2.1 g, 2.52 mmol) in DCM (10.5mL) was stirred at room temperature. Next, 3,3-dimethoxy-1-propene (2.03g, 17.7 mmol) followed by CSA (0.035 g, 0.15 mmol) were added to thesolution. After the solution was stirred for 3.5 hours, LCMS indicatedthe reaction had gone to completion. The reaction was diluted with DCM(25 mL) and added to a separatory funnel with 55 mL 5% NaHCO₃ solution.The layers were separated and the aqueous layer was washed with DCM (25mL). The two organic layers were combined, washed with brine, dried overNa₂SO₄ and concentrated. A flash chromatography column was packed withsilica gel (230-400 mesh) and wet with 50:50 MTBE/heptane (1000 mL). Thereaction mixture was dissolved in MTBE (10 mL), loaded on the column andeluted with 50:50 MTBE/heptane. The fractions were collected, pooled,concentrated and dried in a vacuum oven at 50° C. to give product offormula (3).

To a solution of the compound of formula (12) (0.500 g, 0.4716 mmol) inmethanol, at −19° C. was added 0.2N HCl (1.2 equiv. 2.83 mL) slowly andstirred, The reaction was quenched by the addition of 5% sodiumbicarbonate solution, after stirring the reaction for about 1 hr. Themixture was diluted with ethyl acetate and partitioned. The organiclayer was washed with water, dried (Na₂SO₄) and rotostripped to providecrude compound of formula (13a). The crude product was purified bynormal phase flash chromatography eluting with 15% ethyl acetate inheptane followed by neat ethyl acetate to provide clean product compoundof formula (13a) (0.235 g) To a solution of compound of formula (1.3a)(0.41 g) in DMF (N₅N-dimethylformamide) was added acroleindimethylacetal (3,3-dimethoxy-1-propene, 2 mL) followed by camphorsulfonic acid(0.1 g), The reaction mixture was heated at ˜50° C. and monitoredperiodically by LC-MS analysis for progress of the reaction. Thereaction was judged complete at about 3 hours and quenched by additionof 5% sodium bicarbonate solution. The reaction mixture was diluted withIPAc (isopropyl acetate, 20 mL) and water (20 mL). The mixture wasshaken well and the organic layer partitioned. The aqueous layer wasre-extracted twice with IPAc. The combined IPAc layers were washed withwater and rotostripped to provide the crude product formula (3a) as afoam. A Kromasil column (10OA, 50 cm×2.1 cm column) was conditioned with65:35 n-heptane: waMTBE (1% water and 1% acetic acid in methyl-/-butylether). The crude compound of formula (3a) was loaded as a solution intoluene onto the Kromasil column and eluted with 65:35 n-heptane:waMTBE.The pure fractions containing the product were pooled, neutralized with˜50 mL of 5% sodium bicarbonate solution. The organic layer waspartitioned and evaporated under reduced pressure and dried overnight inthe vacuum oven at 40° C., to provide the pure compound of formula (3a)as a white solid (˜57 mg). The product was characterized by HPLC/LC-MSand high field multidimensional NMR spectroscopy, An alternative methodfor synthesizing the compound of formula (3) is shown below withreference to Schemes 2 to 4.

As shown, paclitaxel of formula 14 is first protected at the 2′-hydroxylwith a hydroxyl protecting group such as tert-butyldimethylsilyl(TBDMS). To a 500 mL round bottom flask (RBF) equipped with a magneticstir bar was charged 50.0 g (58.6 mmol) paclitaxel, formula 14, 14.0 g(205 mmol, 3.5 eq.) imidazole, and 26.5 g (176 mmol, 3.0 eq.) TBDMS-Cl.The flask was placed under a nitrogen environment and 350 mL (7 mL/gpaclitaxel) anhydrous N,N-dimethyl formamide (DMF) was charged to theflask. The reaction was stirred at room temperature for twenty hours,then was worked up by diluting the reaction solution in 600 mL isopropylacetate (IPAc) and washing with water until the aqueous wash reached pH7, then with brin. The organic partition was dried over magnesiumsulfate, filtered and then was evaporated to a white foam solid to yield66.9 g (93.0 area percent) of unpurified 2′-O-TBDMS protected compoundof formula 15.

Next, the 10-acetyl group is removed using methods known in the art,such as by hydrazinolysis. To a 1 L RBF equipped with a magnetic stirbar was charged 59.5 g compound 15 and 600 mL (10 mL/g) IPAc. Thesolution was stirred to dissolve compound 15, then 60 mL (1 mL/g)hydrazine hydrate was charged to the flask and the reaction stirred atroom temperature for one hour. The reaction was worked up by dilutingthe reaction solution in 1.2 L IPAc and washing first with water, thenammonium chloride solution, then again with water until the aqueous washwas pH 7 and lastly with brine. The organic partition was dried overmagnesium sulfate, filtered and evaporated to 55.8 g of solid. The solidwas redissolved in 3:1 IPAc (1% water):heptane to a concentration 0.25g/mL total dissolved solids (TDS) and purified on a YMC silica column;the column eluent was monitored for UV absorbance. The fractions werepooled based on HPLC analysis and evaporated to yield 39.3 g (98.6 areapercent) of the 2′-O-TBDMS-10-deacetyl compound of formula 16.

The 7-hydroxyl is further protected with a protecting group such astriethylsilyl (TES). To a 500 mL RBF equipped with a magnetic stir barwas charged 39.3 g (42.5 mmol) compound 16 and 15.6 g (127 mmol, 3 eq.)4,4-dimethylaminopyridine (DMAP). The flask was placed under nitrogenand 390 mL (10 mL/g) anhydrous dichloromethane (DCM) was charged to theflask to dissolve the solids followed by 14 mL (84.9 mmol, 2 eq.)TES-Cl. The reaction was stirred at room temperature for three hours.The reaction was worked up by evaporating the reaction solution toapproximately half its starting volume and diluting it in 300 mL EtOAcand washing with water and dilute HCl solutions until the pH of theaqueous wash was approximately 7, then washing with brine. The organicpartition was dried over magnesium sulfate and evaporated to yield 42.0g (97.7 area percent) of white solid of formula 17.

Next, oxidation of the 10-hydroxyl yields a 9,10-diketo compound. To a 1L RBF equipped with a magnetic stir bar was charged 41.0 g (39.4 mmol)of the compound of formula 17, 2.1 g (5.92 mmol, 0.15 eq.) oftetrapropylammonium perruthenate (TPAP), 13.9 g (118 mmol, 3 eq.)N-methylmorpholine-N-oxide (NMO). The flask was placed under nitrogenand 720 mL (˜20 mL/g) anhydrous DCM charged to the flask to dissolve thesolids. The reaction was stirred at room temperature for 22 hours. Thereaction was worked up by concentrating the reaction solution to halfits volume and then drying the reaction contents onto 175 g silica gel(EM Sciences 40-63 μ). The product containing silica was placed on 30 gof clean silica gel (EM Sciences 40-63 μ) and the product eluted fromthe silica with 4 L methyl tert-butyl ether (MTBE). The MTBE wasevaporated to yield 37.3 g (93.2 area percent)2′-O-TBDMS-7-O-TES-9,10-diketo compound of formula 18.

Selective reduction of the 9,10-diketo compound yields the9,10-α,α-hydroxy compound. To a 2 L RBF equipped with a magnetic stirbar was charged 37.3 g (35.9 mmol) protected 9,10-diketo compound offormula 18 and 900 mL (˜30 mL/g compound 18) of 3:1 EtOH/MeOH. Thesolution was stirred to dissolve the solids then the flask was placed inan ice/water bath and the solution was stirred for 30 minutes. Then 8.1g (216 mmol, 6 eq.) of sodium borohydride (NaBH₄) was charged to theflask and the reaction stirred in the ice/water bath for five hours. Thereaction was worked up by diluting the reaction solution in 1 L IPAc andwashing with 4×750 mL water, then with 200 mL brine. The organicpartition was dried over magnesium sulfate. The aqueous washes werereextracted with 500 mL IPAc. The organic re-extract solution was washedwith 100 mL brine then dried over magnesium sulfate and combined withthe first organic partition. The IPAc solution was concentrated untilsolids began precipitating out then heptane was added to the solution tocrystallize the product of formula 19. The crystallizing solution wasplaced in a freezer overnight. Three crystallizations were done on thematerial, the first yielded 4.1 g (95.3 area percent) product, thesecond yielded 18.3 g (90.9 area percent) product, and the third yielded2.9 g (81.7 area percent) product. The original work on this reactionemployed flash chromatography to purify the product. However, thecrystallizations that were performed gave similar purity, by HPLC, tothe chromatographed material from earlier work.

To a 25 mL RBF, equipped with a magnetic stir bar and under a nitrogenenvironment, was charged 300 mg (0.288 mmol) of the compound of formula19, (0.720 mmol, 2.5 eq.) acid chloride (CH₃COCl), 140 μL (1.01 mmol,3.5 eq.) triethyl amine (TEA), 13 mg (0.086 mmol, 0.3 eq.) 4-PP, and 10mL anhydrous DCM. The reactions were stirred at room temperature for 15+hours; reactions generally ran overnight and were monitored by TLCand/or HPLC in the morning for consumption of starting material. Thereactions were worked up by diluting the reaction solution in 20 mLEtOAc and washing with water until the pH of the water washes wasapproximately 7. The organic solution was then washed with brine anddried over sodium sulfate before evaporating to dryness. The resultingproduct is the compound of formula 20.

When the reagent used is a carboxyl anhydride, an exemplary procedure isas follows. To a 25 mL RBF, equipped with a magnetic stir bar and undera nitrogen environment, was charged 300 mg (0.288 mmol) of the compoundof formula 19, (2.88 mmol, 10 eq.) acid anhydride (CH₃COOCOCH₃), 106 mg(0.864 mmol, 3 eq.) DMAP, and 5 mL anhydrous DCM. The reactions werestirred at room temperature for 15+ hours. The reactions were worked upby adding 5 mL saturated sodium bicarbonate solution to the reactionflask and stirring for 5 minutes. The solution was then transferred to aseparatory funnel and organics were extracted with 20 mL EtOAc. Theorganic extract was then washed with saturated sodium bicarbonate andwater until the pH of the water washes was approximately 7. The organicpartition was then washed with brine and dried over sodium sulfatebefore evaporating to dryness. The compound of formula 20 may bedeprotected at the 2′- and 7-positions in either a two-step process or asingle step. For example, as shown in Scheme 3, the 7-O-TES group may beremoved from formula 20 to give formula 21 using acetonitrile (ACN) andaqueous HF.

To a 500 mL Teflon bottle equipped with a magnetic stir bar is charged2.50 g (2.40 mmol) of the compound of formula 20 and 100 mL ACN. Thebottle is placed in an ice/water bath and the solution stirred for 30minutes. Next, 0.8 mL of 48% HF aqueous is added slowly to the reactionsolution and the reaction stirred in the ice/water bath for 20 minutes.The reaction is monitored by TLC for disappearance of the startingmaterial. The reaction is worked up by diluting the reaction solution byadding 200 mL EtOAc and quenching the acid by adding 25 mL saturatedsodium bicarbonate solution to the bottle and stirring for 10 minutes.The solution is then transferred to a separatory funnel and the organicpartition washed with water until the pH of the water wash isapproximately 7, then washed with brine. The organic partition is driedover sodium sulfate and then evaporated to a solid of formula 21.

Next, the 2′-O-protecting group may be removed from formula 21 to giveformula 22 as shown in Scheme 3. To a 50 mL Teflon bottle equipped witha magnetic stir bar was charged, 500 mg of the compound of formula 21and 5 mL anhydrous THF. Next, 1 mL HF-pyridine solution was slowlycharged to the reaction solution. The reaction was stirred at roomtemperature for 1 hour; reaction progress was monitored by TLC and/orHPLC for disappearance of starting material. The reaction was worked upby adding 10 mL EtOAc to the bottle to dilute the reaction solution andthen saturated sodium bicarbonate was slowly added to the bottle toneutralize the HF. The solution was transferred to a separatory funneland the organic partition was washed with 10 wt % sodium bicarbonatesolution then water until the pH of the water wash was approximately 7.Then the organic partition was washed with brine and then dried oversodium sulfate before evaporating to a solid of Formula (22). Further,as indicated above, the 2′- and 7-positions of the compound of formula20 may be deprotected in a one-step procedure usingtetrabutylammoniumfluoride (TBAF) to directly produce formula 22. A 10mL RBF equipped with a magnetic stir bar was charged with 100 mg of thecompound of formula 20 and 5 mL EtOAc or THF to dissolve the taxane.Next, 100 μL of 1M TBAF in THF was charged to the flask and the reactionwas stirred at room temperature for 1 hour; the reaction was monitoredby TLC and/or HPLC for disappearance of starting material. The reactionwas worked up by washing the reaction solution with water and thenbrine. The organic partition was dried over sodium sulfate andevaporated to a solid of formula 22. This method removes both the2′-O-TBDMS protecting group and the 7-O-TES protecting group. As shownfor example in Scheme 4, the compound of formula 22 may be protected asa 7,9-acetal, such as a cyclic acetal such as with anisaldehyde dimethylacetal to form a compound of formula 23.

To a 50 mL RBF was charged 1.15 g (1.35 mmol) of the compound of formula22 and 25 mL anhydrous DCM, under nitrogen. 3434 (2.02 mmol, 1.5 eq.)anisaldehyde dimethyl acetal was charged to the flask, followed by 51 mg(0.269 mmol, 0.2 eq.) p-toluenesulfonic acid (PTSA). The reaction wasstirred at room temperature for 45 minutes then was worked up byextracting the product with EtOAc and washing with saturated sodiumbicarbonate solution followed by water. The organic partition wasevaporated to yield approximately 1.5 g of crude product. The crudeproduct was purified by flash chromatography to yield 0.72 g of pureproduct of formula 23.

Next, the side chain is cleaved to form the compound of formula 24. To a25 mL RBF was charged 720 mg (0.740 mmol) of the compound of formula 23and 15 mL anhydrous THF, under nitrogen. The flask was placed in anice/water/ammonium chloride, −13° C. bath. Solid lithium borohydride(29.0 mg, 1.33 mmol, 1.8 eq.) was charged to the reaction flask and thereaction stirred at −13° C. for two hours before raising the temperatureto 0° C. The reaction was worked up after five hours fifteen minutes bydiluting with EtOAc and washing with water and ammonium chloridesolution. The organic partition was evaporated to yield 650 mg of crudecompound but HPLC indicated that there was only approximately 20%product and mostly unreacted starting material; therefore, the reactionwas restarted by repeating the above procedure and running the reactionfor an additional six hours. The organic partition was evaporated toyield approximately 660 mg of crude product. The compound was purifiedon a spherical silica column to yield the compound of formula 24.

The compound of formula 24 is then coupled with formula 25 to providethe compound of formula 26. To a 5 mL RBF was charged 180 mg (0.255mmol) of the compound of formula 24 and 105 mg (0.510 mmol, 2.0 eq.)DCC. Toluene (2 mL) was then added to dissolve the solids. Next, formula28 (158 mg, 0.383 mmol, 1.5 eq.) was dissolved in 1.0 mL DCM and thesolution was charged to the reaction flask followed by 6 mg (0.038 mmol,0.15 eq.) 4-PP. The reaction was stirred at room temperature for 23hours and then was quenched by adding 11.5 μL acetic acid and 4 μL waterand stirring for one hour. MTBE was added to the reaction flask toprecipitate DCU and the reaction solution was filtered to remove theprecipitate. The filtrate was slurried with activated carbon then passedacross a silica plug to remove the 4-PP salts. The eluent was evaporatedto a solid to yield 271 mg of crude coupled product of formula 26.

The 7,9-acetal and N,O-acetal protecting groups may then be removed andan N-acyl group added to form the compounds of formula 27 and 28, whichmay be separated from each other by liquid chromatography or kepttogether for the next step. While the same anisaldehyde group is used atboth the 7,9-acetal and N,O-acetal in the exemplary compound of formula26, such that both groups may be removed in a single step, it should beappreciated that other acetal protecting groups are contemplated suchthat multiple deprotection steps may be required. To a 10 mL RBF wascharged, 270 mg (0.245 mmol) of the compound of formula 26, 220 mg (0.8g/g coupled ester) Degussa type palladium on carbon, and 4.1 mL THF. Ina separate vial, 99 μL conc. HCl was diluted in 198 μL water and 1.0 mLTHF. This solution was added to the reaction flask and the flask wassealed and placed under hydrogen. The hydrogenation reaction was stirredfor 31 hours then was quenched by removing the hydrogen and filteringthe catalyst from the reaction solution then adding 84.5 μL (0.368 mmol,1.5 eq.) t-butoxy carbonyl (t-BOC) anhydride followed by 684 μL TEA. Thereaction stirred an additional 21 hours and then was worked up, dilutingthe filtrate with EtOAc and washing with water. The organic partitionwas evaporated to approximately 370 mg of oil. The oil was purifiedfirst by flash chromatography, then preparative TLC (PTLC) then by asemi-prep reverse phase column to yield 3.9 mg of pure product offormula 27 and 28.

A 7,9-acetal may then be formed to provide the compound of formula 3. Ina HPLC vial insert, 3.4 mg (4.13 μmol) of the compounds of formula 27and 28 was charged followed by 70 μL DCM. Next, 12.8 μL of a 1 to 20diluted acrolein dimethyl acetal in DCM (0.64 μL acetal, 5.37 μmol, 1.3eq.) was charged to the insert followed by 8.4 μL (0.413 μmol, 0.1 eq.)of a 0.05M PTSA solution in DCM. The reaction was lightly agitated thensat at room temperature. The reaction took more additions of the acetalsolution to drive it to completion then was worked up after a couple ofdays by filtering the solution through approximately 80 mg of basicactivated alumina. The alumina was washed with DCM then EtOAc and thefractions evaporated to dryness. The crude compound was purified on anormal phase analytical column to yield 605 μg of the compound offormula 3. The compound of formula 3 may then be separated into itsindividual diastereoisomers to produce the compound of formula S-(1) viachromatography using a solvent composition of 35% MTBE in heptane asdescribed in the examples.

As generally described and specifically exemplified above, theseprocesses may be performed with the isolation of one or more of theintermediate compounds, or the process may be performed without theisolation and purification at each and every single processing steps.

Standard procedures and chemical transformation and related methods arewell known to one skilled in the art, and such methods and procedureshave been described, for example, in standard references such asFiesers' Reagents for Organic Synthesis, John Wiley and Sons, New York,N.Y., 2002; Organic Reactions, vols. 1-83, John Wiley and Sons, NewYork, N.Y., 2006; March J. and Smith M.: Advanced Organic Chemistry,6^(th) ed., John Wiley and Sons, New York, N.Y.; and Larock R. C.:Comprehensive Organic Transformations, Wiley-VCH Publishers, New York,1999.

Accordingly, the invention additionally provides a process for thepreparation of the compound of formula S-(1), which comprises separationof the S-(1) isomer from a mixture with the R-(1) isomer bychromatography. The invention additionally provides a process for thepreparation of the compound of formula S-(1), which comprises separationof the S-(1) isomer from a mixture with the R-(1) isomer and thecompound of formula (2) by chromatography. Preferably, the compound offormula S-(1) is prepared by reaction of the compound of formula (13)with 3,3-dimethoxy-1-propene to give the acetal compound of formula (3)followed by isolation of the compound of formula S-(1) bychromatography. The chromatography is preferably conducted using silicagel as herein described.

An alternative method for synthesizing the compound of formula (1) isshown in Scheme 5.

A preferred method for synthesizing the compound of formula (1) is shownin Scheme 6.

Another preferred method for synthesizing the compound of formula (1) isdetailed below with reference to Scheme 7.

I. Oxidation of 10 DAB III, Formula (4):

A 4 L reaction flask, rinsed with dried EtOAc (300 mL) and held underN₂, was charged with dried EtOAc (1250 mL). Agitation was begun anddried (4) (100 g, 0.184 mol) was added. The addition of USP EtOH (800mL) followed and the reaction mixture was cooled to −1.3° C. (internaltemperature). Anhydrous CuCl₂ (86.4 g, 3.5 eq) was added and solids fromthe sides of the flask were washed into the mixture with anhydrous EtOH(450 mL). The reaction mixture was cooled to ≤−13° C. and anhydrous TEA(90 mL, 3.5 eq) was added slowly. The reaction was monitored byHPLC/TLC. At 1 h the reaction was judged complete (<5% (4)).

TFA (36 mL) was added to quench the reaction and stirring continued for15 min. The reaction mixture was transferred to a 10 L rotovap flask.EtOAc (500 mL) and EtOH (300 mL) were added to the reaction flask,stirred for 2 min and the rinse added to the contents of the rotovapflask, which was evaporated on the rotovap at 40° C. until no furtherdistillation occurred (80 min). Acidified ethanol (300 mL) was added tothe residue and the resulting slurry was transferred to a 2 L rotovapflask. The first rotovap flask was rinsed into the second with acidifiedEtOH (400 mL). Again, the mixture was evaporated on the rotovap at 40°C. until no further distillation occurred (1 h). Acidified ethanol (305mL) was added to the rotovap flask and the mixture was stirred on therotovap at 40° C. for 10 min. The contents of the flask were then cooledto 5° C. and filtered. The rotovap flask was rinsed (2×) with cold (2°C.) acidified ethanol (300 mL) and the rinse was transferred completelyto the filter to wash the solids. The solids were dried in the vacuumoven overnight at 45° C. to give (29a). HPLC Area %=91.3%. Yield=96.72g.

II. Tesylation of (29a) to form (30):

To (29a) (96.72 g, 0.1783 mmol) in a 10 L rotovap flask was added ethylacetate (3000 mL, 30 mL/g). The solution was evaporated on the rotovapat 40° C. to approximately half the original volume (distilledvolume=1680 mL). Toluene (1000 mL, 10 mL/g) was added to the remainingsolution and it was evaporated on the rotovap at 40° C. until solidswere obtained (45 min). The solids were suspended in toluene (1000 mL,10 mL/g) and the suspension was evaporated on the rotovap at 40° C. (˜1h) to dry solids. The solids were transferred to a 2 L flask equippedwith a mechanical stirrer, thermocouple, addition funnel and N₂ stream(previously purged for 5 min). The solids in the rotovap flask wererinsed into the reaction flask with anhydrous pyridine (292 mL, 3 mL/g)and agitation was begun. Upon dissolution, agitation was continued andthe contents of the flask were cooled to −20° C. Triethylsilyltrifluoromethanesulfonate (120.9 mL, 3.0 eq) was slowly added to thereaction mixture to maintain the internal temperature of the reaction at≤−10° C. After the addition of TES-OTf was complete, the reactionmixture was allowed to warm to −5.8° C. and agitation continued. Thirtyminutes after the addition of TES-OTf, sampling was begun and continuedat thirty-minute intervals for HPLC/TLC. The reaction was judgedcomplete at 2 h when HPLC/TLC indicated <2% mono-TES derivativeremaining.

The reaction mixture was cooled to −17.5° C. Methanol (19.3 mL, 0.2mL/g) was added to quench the reaction and the reaction mixture wasstirred for 5 min. While allowing the mixture to warm to ambienttemperature, MTBE (500 mL) was slowly added with stirring and themixture was transferred to a separatory funnel. Residues remaining inthe reaction flask were washed into the separatory funnel withadditional MTBE (200 mL, 2 mL/g), then water (250 mL, 2.5 mL/g) andsaturated NH₄Cl solution (250 mL, 2.5 mL/g) were added. The mixture wasagitated and the layers were separated. The organic layer wastransferred to a clean container. MTBE (250 mL, 2 mL/g) was added to theaqueous layer. It was agitated and the layers were separated. The secondorganic layer was washed into the first organic layer with MTBE (100 mL)and water (200 mL, 2 mL/g) was added to the combined layers. Thismixture was agitated and the layers were separated. The organic layerwas transferred to a 2 L rotovap flask and evaporated to a residue at40° C. n-Heptane (500 mL, 5 mL/g) was added to this residue and thesolution was again evaporated to a residue at 40° C. n-Heptane (1000 mL,˜10 mL/g) was added again and the solution was evaporated to one-half ofits volume (distilled volume=375 mL). n-Heptane (300 mL, ˜2.5 mL/g) wasadded and the solution was stirred for 35 min on the rotovap at 40° C.The solution was then cooled to −15.7° C. while stirring was continuedfor ˜2.5 h. The solution was filtered. The solids remaining in the flaskwere rinsed into the filtration funnel with cold (<5° C.) n-heptane (100mL) and all the solids were collected and dried overnight in the vacuumoven to give 111.2 g (30). HPLC Area % purity=93.4%.

III. Reduction of (30) to Prepare(31):

To a stirred solution of THF (560 mL, 5 mL/g) under N₂ in a 4 L reactionflask, was added (30) (111 g, 0.144 mol,) followed by anhydrous ethanol(560 mL, 5 mL/g). The mixture was stirred to dissolve the solids andthen cooled to −12° C. 2 M LiBH₄ in THF (72 mL) was added slowly tocontrol the reaction temperature (temp=−11.9 to −9.7° C.). The reactionmixture was stirred and sampled for HPLC/TLC at 30 min intervals.Additional 2 M LiBH₄ in THF was introduced slowly (72 mL, 1.0 eq) to thereaction flask (temp=−9.6° C. to −7.1° C.) and agitation continued for30 min. A third addition of 2 M LiBH₄ in THF (36 mL, 0.5 eq) was made inthe same manner as the previous additions (temp=−7.6° C. to −6.7° C.),but with the bath temperature adjusted to 15° C. following the additionof the LiBH₄ solution and to 12.5° C. ten minutes later. At 1 hfollowing the final LiBH₄ addition, the reaction was judged complete(mono reduced product≤3% relative to (31)).

The reaction mixture was cooled to −10.8° C. and 10% ammonium acetate inEtOH (560 mL) was added slowly and cautiously to allow the foam tosettle and to control the temperature of the solution≤−3° C. Thereaction mixture was transferred to a 2 L rotovap flask and any residuesin the reaction flask were rinsed into the rotovap flask with EtOH (250mL) and the contents of the rotovap flask were evaporated on the rotovapat 40° C. to an oil. Methanol (560 mL) was added to the residue. Water(1700 mL) was added to a 5 L flask equipped with an addition funnel andmechanical stirrer and was vigorously agitated. To precipitate theproduct, the methanol solution of the reaction mixture (748 mL) wasslowly added to the flask containing water. The resulting mixture wasfiltered and the solids were washed with water (650 mL). A portion ofthe water was used to wash solids remaining in the precipitation flaskinto the filtration funnel. The solids were placed in the vacuum ovenovernight at 45° C. to give 139.5 g of slightly wet non-homogeneousproduct, (31). HPLC area % purity=92.8%.

IV. Acetylation/Deprotection of (31) to Prepare (33):

Acetylation: To (31) (138 g, 0.178 mol) in a 2 L rotovap flask was addedIPAc (1400 mL, 10 mL/g). The solution was evaporated on the rotovap at40° C. to an oil. The procedure was repeated. Dried IPAc (550 mL) wasthen added to the residual oil and the contents of the rotovap flaskwere transferred to a 1 L reaction flask, equipped with a mechanicalstirrer, addition funnel, thermocouple and a N₂ stream. The rotovapflask was washed into the reaction flask with IPAc (140 mL). DMAP (8.72g, 0.4 eq), anhydrous TEA (170 mL, 7 eq) and acetic anhydride (100.6 mL,6 eq) were added to the contents of the reaction flask and the mixturewas stirred and heated to 35° C. While continuing agitation and heatingto 35° C., the reaction was monitored by HPLC/TLC at 1-hour intervals.

Upon completion of the reaction, as indicated by the absence of (31) (3h total time), the reaction mixture was cooled to 19.7° C. and saturatedammonium chloride solution (552 mL) was added. After stirring for 15min, the mixture was transferred to a separatory funnel, the layers wereseparated and the aqueous layer was removed. Water (280 mL) was added tothe organic layer and the mixture was stirred for 4 min. The layers wereagain separated and the aqueous layer was removed. The organic layer wastransferred to a 2 L rotovap flask and the remaining content of theseparatory funnel was washed into the rotovap flask with IPAc (200 mL).The mixture was evaporated to dryness on the rotovap at 40° C. to give˜124 g (32) as pale yellow oily foam.

Deprotection: To the rotovap flask containing (32) (124 g) was addedmethanol (970 mL, 7 mL/g). Sampling for HPLC/TLC was begun and continuedat 1-hour intervals. The (32)/methanol solution was transferred to a 3 Lreaction flask and agitation was begun. The remaining content of therotovap flask was washed into the reaction flask with methanol (400 mL).Acetic acid (410 mL, 3 mL/g) and water (275 mL, 2 mL/g) were added andthe reaction mixture was heated to 50° C. and stirred. With thetemperature maintained between 50° C. and 55° C., the reaction wasmonitored by HPLC/TLC at 1-hour intervals for the disappearance of thestarting material, formation and disappearance of the mono-TESintermediate and formation of the product, (33).

Upon completion (˜9 h), the reaction mixture was cooled to rt andtransferred to a 10 L rotovap flask. Solvent exchanges to n-heptane(2×1370 mL, 1×1000 mL) and IPAc (2×1370 mL, 1×1500 mL) were performed.IPAc (280 mL, 2 mL/g) and silica (140 g, 1 g/g) were added to therotovap flask and the contents were evaporated on the rotovap at 40° C.until no further distillation occurred and free flowing solids wereobtained. The dry silica mixture was loaded onto a silica pad (7 cmcolumn, 280 g silica), conditioned with 2:1 n-heptane/IPAc (500 mL, 2mL/g silica) and washed (4×) with 2:1 n-heptane/IPAc, 2 mL/g silica,3400 mL total) and (4×) with 1:1 n-heptane/IPAc (3020 mL total, 2 mL/gsilica) until all impurities were removed as indicated by TLC. Each wash(˜840 mL) was collected as a separate fraction and analyzed by TLC. Thesilica pad was then washed (5×) with waEtOAc (1% water, 1% AcOH inEtOAc) (3950 mL total, 2 mL/g silica) and with 1:1 MeOH/EtOAc and eachwash (˜840 mL) was collected as a separate fraction. The product elutedwith fractions 11-15. The fractions containing (33) as indicated byHPLC/TLC were combined, transferred to a rotovap flask and evaporated todryness on the rotovap at 40° C. The residue in the flask was dissolvedand evaporated to dryness: first with IPAc (1055 mL) and n-heptane (550mL) and a second time with IPAc (830 mL) and n-heptane (410 mL). IPAc(500 mL) was then added to the residue, the solution was transferred toa 2 L round bottom flask and n-heptane (140 mL) was added. The resultingsolution was evaporated on the rotovap and dried in the vacuum oven at40° C. to give (33) as foam. To dissolve the foam, IPAc (160 mL) wasadded to the flask followed by toluene (800 mL). The solution wasevaporated on the rotovap under vacuum at 50° C. until half of thesolvent was removed and solids were forming. The contents of the flaskwere stirred and cooled to 21° C. for 1.5 h. The solids were filtered ina 90 cm filtration funnel on #54 Whatman filter paper and were washedwith toluene (165 mL), transferred to the vacuum oven and dried at 40°C. to give 62.6 g of (33). HPLC area %=96.9%

VI. Acetal Formation: (33) to (34)

To a 3 L reaction flask containing (33) (25 g, 42.4 mmol) was addedtoluene (375 mL) and the reaction mixture was cooled to ˜−15° C. TFA(9.8 mL, 3.0 eq) was slowly added. This was followed by the addition ofacrolein diethyl acetal (8.7 g) and the reaction was monitored by HPLCuntil <3% of (33) remained.

Hydrated silica was prepared by mixing silica (25 g) and water (25%) anda “basified silica” mixture was prepared by mixing a solution of K₂CO₃(17.6 g, 3.0 eq) in water (1 mL/g (33)) with 50 g silica. Upon reactioncompletion, the hydrated silica was added to the reaction mixture and itwas stirred for 30 to 45 min while maintaining the temperature≤5° C. Thebasified silica was then added to the mixture while continuing tomaintain the temperature≤5° C. and the pH>5. After stirring for ˜15 min,the mixture was filtered. The silica was washed with ˜20 mL/g tolueneand the filtrates were combined and concentrated. The residue wasdigested with 1 mL/g toluene for ˜4 h. The resultant solids werefiltered and washed with 80:20 toluene/heptane to give 25 g of (34).HPLC area %=98%. Mass yield=66%.

VII. Preparation of Compound (1) from (34):

To THF (300 mL, 8 mL/g) stirring in a 1 L reaction flask (rinsed withTHF (500 mL)) was added (34) (35.7 g, 0.0570 mol). Purified (35a) (30.9g, 1.25 eq) was added to the reaction mixture followed by the additionof NMM (11.5 mL, 1.8 eq), DMAP (2.77 g, 0.4 eq) and THF (75 mL, 2 mL/g).The mixture was stirred while N₂ was bubbled from the bottom of theflask to mix and dissolve the solids. Pivaloyl chloride (11.5 mL, 1.6eq) was then added slowly to the reaction mixture. The reaction mixturewas warmed and the temperature maintained at 38° C.±4° C. while stirringcontinued and N₂ continued to be bubbled from the bottom of the flask.The reaction mixture was analyzed by HPLC/TLC for consumption ofstarting material and formation of the coupled ester, (36a), at 30 minintervals beginning 30 min after the addition of the pivaloyl chloride.

After 1 h the reaction was judged complete and the reaction mixture wascooled to 2° C. 0.5 N HCl in MeOH (280 mL, ˜20 mL/mL NMM) was added tomaintain the pH of the reaction mixture=1.5-1.9. The reaction mixturewas stirred at 2° C.±2° C. and monitored by HPLC/TLC at 30 min intervalsfor consumption of (36a) and formation of (1) and the acrolein acetalhydrolyzed by-product. Upon completion at 2 h the reaction was quenchedwith 5% aqueous sodium bicarbonate (300 mL) and IPAc (185 mL, 5 mL/g)was added. The reaction mixture was transferred to a 2 L rotovap flaskand the reaction flask rinsed into the rotovap flask 2× with 60 mL IPAc.The mixture was evaporated under vacuum at 40° C. until a mixture of oiland water was obtained. IPAc (200 mL) was added to the oil and watermixture and the contents of the flask were transferred to a separatoryfunnel. The reaction flask was rinsed into the separatory funnel withIPAc (100 mL) and the contents of the separatory funnel were agitatedand the layers were separated. The aqueous layer was removed. Water (70mL) was added to the organic layer and, after agitation, the layers wereseparated and the aqueous layer was removed. The organic layer wastransferred to a rotovap flask and evaporated under vacuum at 40° C. toa foam, which was dried in the vacuum oven to give 64.8 g crude (1).HPLC area %=45.5%.

VIII. Purification Procedures:

Normal Phase Chromatography: The 6″ Varian DAC column was packed withKromasil (5 Kg, 10 μm, 100 Å normal phase silica gel). The 50-cm bedlength provided a 9 L empty column volume (eCV). The column had beenregenerated (1 eCV 80:20 waMTBE:MeOH) and re-equilibrated (1 eCV waMTBE,1 eCV 65:35 n-heptane:waMTBE).

The crude (1) (64.70 g), was dissolved in MTBE (180 mL) and heated to−40° C. n-Heptane (280 mL) was slowly added to the solution. This loadsolution was pumped onto the column using a FMI “Q” pump. The column wasthen eluted with 65:35 n-heptane:waMTBE at 800 mL/min. A 34 L forerun(˜3.8 eCV) was collected followed by 24 fractions (500 mL each).Fractions 1 through 23 were combined and concentrated to dryness on arotovapor. The residue was dried in the vacuum oven overnight to provide41.74 g (1). HPLC area %=99.4%.

Final Purification: The normal phase pool was dissolved in USP EtOH (6mL/g) and concentrated to dryness three times. The resultant residue wasdissolved in USP EtOH (2 mL/g). This ethanolic solution was slowly addeddrop-wise to water (deionized, 20 mL/g) with vigorous stirring. Theresultant solids were vacuum filtered and washed with cold DI water. Thesolids were dried in the vacuum oven at 40° C. overnight to give 38.85 g(1). HPLC area %=99.5%.

IX. Coupling of (35b) to (34) to Form (1):

Anhydride/Coupling (35b) with (34): A 10 mL round bottom flask with twonecks was heated to eliminate water, then allowed to cool under N₂atmosphere. To the flask was added (34) (125 mg, 0.2 mmol), THF (1.25mL), 4-methylmorpholine (40 μL, 0.36 mmol), DMAP (10.9 mg, 0.009 mmol),(35b) sodium salt (110 mg, 0.254 mmol) and finally trimethylacetylchloride (40 μL, 0.319 mmol). The reaction mixture was stirred at 40° C.under N₂. After about 2 hours, additional 4-methylmorpholine (11 μL,0.01 mmol), (35b) sodium salt (41 mg, 0.1 mmol) and trimethylacetylchloride (13 μL, 0.1 mmol) were added to assist formation of theanhydride intermediate which then coupled to (34). After about 2additional hours, 4-methylmorpholine (11 μL, 0.010 mmol),trimethylacetyl chloride (13 μL, 0.104 mmol) and (35b) sodium salt (42mg, 0.1 mmol) were added. After 1.5 hours more, the reaction was placedinto a freezer at −20° C. overnight. The following morning, stirring wasresumed and the reaction was heated to 45° C. for 2 hours. Additional4-methylmorpholine (22 μL, 0.02 mmol) and trimethylacetyl chloride (25μL, 0.201 mmol) were added. An additional 2 hours of stirring resultedin the reaction reaching ˜90% completion.

To quench, the reaction mixture was removed from heat and allowed tocool to RT with stirring, and MTBE (2 mL) was added followed by water (1mL). The mixture was partitioned and the organic phase was washed withbrine (40 μL). The organic phase was concentrated at 40° C. to obtaincrude product as a pink foam. The pink foam was dissolved into MTBE (500μL) and added dropwise to stirring n-heptane (5 mL) at ˜−20° C. to givepink precipitate. The mixture was vacuum filtered and the solids weredried overnight in a vacuum oven at 40° C. to yield the desired coupledester (82 mg), as indicated by LC/MS. The coupled ester (36b) waspurified by flash chromatography on normal phase silica, eluting with anIPAc/n-heptane system of increasing polarity. Approximately 26 mg of thepurified coupled ester (36b) was recovered as confirmed by LC/MS.

Deprotection of (36b) to Form (1):

The coupled ester (36b) (15 mg, 0.001 mmol) was dissolved into THF (1mL). A 250 μL aliquot of the solution was diluted 1:1 with THF. Thesolution was stirred on an ice bath at ˜0° C., after which HCl (0.5 N inMeOH, 25 μL) was added. The reaction was monitored by LC/MS, whichindicated the formation of (1).

Methods for the preparation of the compounds of formula 35a and 35b andfurther suitable coupling conditions for converting the compound offormula (34) into the compound of formula (1) are described in WO2007/126893.

Chemical Example 1. Separation of Diastereoisomers of Formula (3) ByNormal Phase Chromatography

A solution of the compound of formula (3), which comprises a mixture ofdiasteroisomers of formula (1) and formula (2) (570 mg) was concentratedto light yellow oil, dried in the vacuum oven for 15 min andre-dissolved in 35:65 MTBE/n-heptane. The solution was loaded onto aflash chromatography column packed with spherical silica (YMC-1701, 56g), which had been conditioned with 35:65 MTBE/n-heptane. The solutionflask was rinsed (2×) with ˜2 mL of MTBE onto the column. The column waseluted with 35:65 MTBE/n-heptane and fractions (25 mL) were collected.Fractions containing the pure product (fractions 23-25) as indicated byvisual spotting (to identify the elution of UV active material) and byTLC analysis (50:50 MTBE/n-heptane) were collected, pooled andconcentrated to give 305 mg of the diastereoisomer of formula S-(1) as awhite solid.

The compound S-(1) was characterized by NMR, including ¹H, ¹³C, HMBC,HSQC, NOESY, COSY and gHSQMBC. The compound of formula S-(1) was alsoanalyzed by β-tubulin binding modeling studies. Similarly compound R-(1)was also characterized by NMR, including ¹H, ¹³C, HMBC, HSQC, NOESY,COSY and gHSQMBC.

Additionally, the invention additionally provides a process for thepreparation of the compound of formula S-(34), which comprisesseparation of the S-(34) isomer from a mixture with the R-(34) isomer bychromatography.

Properties and Uses of the Compounds of the Invention

The present application provides various methods of treating abnormalcell growth in a mammal, such as a human, by administering to the mammala therapeutically effective amount of a composition comprising thecompound of formula S-(1) or pharmaceutically acceptable salts, solvatesor hydrates thereof.

In one aspect of the above method, the composition comprising thecompound of formula S-(1) is administered via parenteral administration.In another aspect of the above method, the parenteral administration isvia intravenous injection or infusion. In another aspect of the abovemethod, the parenteral administration is for about 60 minutes or less.In another aspect of the above methods, the administration of theintravenous infusion is once every 7 days for 3 weeks, followed by a 7day rest period, in a 28 day cycle, for at least one cycle. In oneparticular variation of the above methods, the administration of theintravenous infusion is once every 21 days, in a 21 day cycle, for atleast one cycle.

In one aspect of the methods above, the intravenous infusion comprisesthe compound of formula S-(1) at a dose of 240 mg/m² or less, or 185mg/m² or less. In another aspect of the methods above, the intravenousinfusion comprises the compound of formula S-(1) at a dose of 185 mg/m²or less, or 160 mg/m² or less. Such amounts can often be reflected inunit doses of about 200 mg to 400 mg, for example about 250 mg, 300 mgor 350 mg.

In one aspect of any of the above-mentioned embodiments of theinvention, the composition comprising the compound of formula S-(1)comprises a polyethoxylated castor oil formulation.

Preferably, the polyethoxylated castor oil formulation is about 50%ethanol and about 50% polyethoxylated castor oil or about 85% ethanoland about 15% polyethoxylated castor oil.

In one embodiment of the application, the composition comprising thecompound of formula S-(1) may be administered parenterally, orally,subcutaneously, or intraperitoneally. In another embodiment of any ofthe above-mentioned embodiments of the invention, the compositioncomprising the compound of formula S-(1) further comprises one or morepharmaceutically acceptable, inert or physiologically active excipients,diluents or adjuvants selected from the group consisting of phomopsin,dolastatin, bevacizumab (Avastin™), steganacin, paclitaxel, taxotere,vinblastine, vincristine, vindesine, vinorelbine, navelbine, colchicine,maytansine, ansamitocin, Iressa, Tarceva, Herceptin, lapatinib,vandetanib, Sorafenib, BAY-57-9006, bevacizumab, cetuximab, gemtuzumab,panitumumab, rituximab, tositumomab, trastuzumab, apolizumab,oregovomab, mitumomab, alembuzumab, ibritumomab, vitaxin, SU-6668,semaxanib, sunitinib malate, SU-14813, vandetanib, Recentin, CP-547632,CEP-7055, AG-013736, pazopanib, combretastatin, squalamine, combrestatinA4 phosphate, TNP-470, neovastat, dasatinib, imatinib, nilotinib,sorafenib, sunitinib, triethylenethiophosphoramine, alitretinoin,altretamine, arsenic trioxide, asparaginase, bexarotene, denileukindiftitox, hydroxycarbamide, masoprocol, mitotane, pegaspargase,tretinoin, raltitrexed, IL-10, IL-12, bortezomib, leuprolide, interferon□, pegylated interferons, atrasentan, melphalan, cyclophosphamide,chlormethine, chlorambucil, trofosfamide, ifosfamide, nitromin,busulfan, thiotepa, chlorambucil, CC-1065, temozolomide, pipobroman,dacarbazine, mechlorethamine, procarbazine, uramustine, RSU-1069,CB-1954, hexamethylmelamine, cisplatin, carboplatin, oxaliplatin,BBR3464, satraplatin, tetraplatin, iproplatin, amsacrine, netropsin,pibenzimol, mitomycin, duocarmycin, dactinomycin, distamycin,mithramycin, chromomycin, olivomycin, anthramycin, bleomycin,liblomycin, rifamycin, actinomycin, adramycin, trichostatin A,propamidine, stilbamidine, rhizoxin, nitroacridine, geldamycin, 17-AAG,17-DMAG, plicamycin, deoxycoformycin, levamisole, daunorubicin,doxorubicin, epirubicin, idarubicin, mitroxantrone, valrubicin,carmustine, fotemustine, lomustine, streptozocin, gemcitabine,5-fluorouracil (5-FU), fludarabine, cytarabine, capecitabine,mercaptopurine, cladribine, clofarabine, thioguanine, pentostatin,floxuridine, pentostatin, aminopterin, methotrexate, pemetrexed,camptothecin, irinotecan, topotecan, epipodophyllotoxin, etoposide,teniposide, aminogluthetimide, anastrozole, exemestane formestane,letrozole, fadrozole, aminoglutethimide, leuprorelin, buserelin,goserelin, triptorelin, abarelix, estramustine, megestrol, flutamide,casodex, anandron, cyproterone acetate, finasteride, bicalutamide,tamoxifen or its citrate salt, droloxifene, trioxifene, raloxifene orzindoxifene, a derivative of 17-β-estradiol such as ICI 164, ICI 384,ICI 182, ICI 780, testolactone, fulvestrant, toremifene, testosterone,fluoxymesterone, dexamethasone, triamcinolone, dromostanolonepropionate, megestrol acetate, methyltestosterone, chlorotrianisene,hydroxyprogesterone, medroxyprogesterone acetate, reloxafine,etanercept, thalidomide, revimid (CC-5013), aziridoquinones,misonidazole, NLA-1, RB-6145, misonidazole, nimorazole, RSU-1069,SR-4233, porfimer, photofrin, verteporfin, merocyanin 540, tinetiopurpurin, PUVA, aminolevulinic acid, methyl aminolevulinate,minodronate, zoledronic acid, ibandronate sodium hydrate or clodronatedisodium, misonidazole, misonidazole, amifostene, oblimersen, TIMP-1 orTIMP-2, marimastat, TLK-286 and mixtures thereof.

In a preferred embodiment, the one or more pharmaceutically acceptableadjuvants is temozolomide. In another preferred embodiment, the one ormore pharmaceutically acceptable adjuvants is bevacizumab (Avastin™).

The compound of the invention may be useful in the treatment of diseaseswhen used alone or in combination with other therapies. For example,when used for the treatment of cancer, the compounds of the inventionmay be administered alone or in combination with radiotherapy, surgicalremoval, hormonal agents, antibodies, antiangiogenics, COX-2 inhibitors,and/or other chemotherapeutic agents such as taxanes, temozolomide,cisplatin, 5-fluorouracil, taxotere, gemcitabine, topoisomerase IIinhibitor, topoisomerase I inhibitor, tubulin interacting agent,antibodies, antiangiogenics, COX-2 inhibitors, hormonal agent,thymidilate synthase inhibitor, anti-metabolite, alkylating agent,farnesyl protein transferase inhibitor, signal transduction inhibitor,EGFR kinase inhibitor, antibody to EGFR, C-abl kinase inhibitor,hormonal therapy combination and aromatase combination.

The compound of the invention may be useful in the treatment of diseaseswhen used alone or in combination with other chemotherapeutics. Forexample, when used for the treatment of cancer, the compounds of theinvention may be administered alone or in combination with aromataseinhibitors, antiestrogen, anti-androgen, a gonadorelin agonists,topoisomerase 1 inhibitors, topoisomerase 2 inhibitors, microtubuleactive agents, alkylating agents, anthracyclines, corticosteroids,IMiDs, protease inhibitors, IGF-1 inhibitors, CD40 antibodies, Smacmimetics, FGF3 modulators, mTOR inhibitors, HDAC inhibitors, IKKinhibitors, P38MAPK inhibitors, HSP90 inhibitors, akt inhibitors,antineoplastic agents, antimetabolites, platin containing compounds,lipid- or protein kinase-targeting agents, protein- or lipidphosphatase-targeting agents, anti-angiogentic agents, agents thatinduce cell differentiation, bradykinin 1 receptor antagonists,angiotensin II antagonists, cyclooxygenase inhibitors, heparanaseinhibitors, lymphokine inhibitors, cytokine inhibitors, bisphosphanates,rapamycin derivatives, anti-apoptotic pathway inhibitors, apoptoticpathway agonists, PPAR agonists, inhibitors of Ras isoforms, telomeraseinhibitors, protease inhibitors, metalloproteinase inhibitors,aminopeptidase inhibitors, thymidilate synthase inhibitors, a DNA crosslinking agents, topoisomerase I or II inhibitors, DNA alkylating agents,ribonuclase reductase inhibitors, cytotoxic factors, and growth factorinhibitors.

In one aspect of the invention the composition comprising the compoundof formula S-(1) further comprises one or more pharmaceuticallyacceptable, inert or physiologically active diluents or adjuvantsselected from the group consisting of cytostatic agent, cytotoxic agent,taxane, topoisomerase II inhibitor, topoisomerase I inhibitor, tubulininteracting agent, antibodies, antiangiogenics, COX-2 inhibitors,hormonal agent, thymidilate synthase inhibitor, anti-metabolite,alkylating agent, farnesyl protein transferase inhibitor, signaltransduction inhibitor, EGFR kinase inhibitor, antibody to EGFR, C-ablkinase inhibitor, hormonal therapy combination and aromatasecombination.

In another aspect of the invention, the composition comprising thecompound of formula S-(1) may be administered alone or in combinationwith one or more anti-cancer therapies. In another aspect of theinvention, the one or more anti-cancer therapies is a chemotherapeuticagent. In another aspect of the invention, the one or more anti-cancertherapies is radiation therapy.

In another embodiment of the invention, the one or more anti-cancertherapies is surgical removal of a tumor. In one aspect of theabove-mentioned embodiments of the invention, the composition comprisingthe compound of formula S-(1) may be administered as a single infusiononce every 21 days in a 21 day cycle. In another aspect of theinvention, the composition comprising the compound of formula S-(1) maybe administered as a single infusion once every seven days, followed bya rest week, in a 28 day cycle. Dose levels on this schedule may exceed185 mg/m² per infusion, with chemo-naive patients tolerating greaterdosing levels, but even in heavily pretreated patients, dosing of 126mg/m² or 150 mg/m² is well tolerated.

In one embodiment of the invention, the composition comprising thecompound of formula S-(1) may be administered before the one or morechemotherapeutic agents. In one aspect of the invention, the compositioncomprising the compound of formula S-(1) may be administered after theone or more chemotherapeutic agents. In another aspect, the compositioncomprising the compound of formula S-(1) may be administered before theradiation therapy. In yet another aspect, the composition comprising thecompound of formula S-(1) may be administered after the radiationtherapy.

In one particular aspect of the above-mentioned embodiments of theinvention, the composition comprising the compound of formula S-(1) maybe administered before the surgical removal. In another aspect, thecomposition comprising the compound of formula S-(1) may be administeredafter the surgical removal.

The application also provides compositions for and a method ofpotentiating the therapeutic benefit of a multi-drug chemotherapeuticregimen, wherein one of the drugs in the regimen comprises the compoundof formula S-(1), by administering the compound of formula S-(1) as anintravenous formulation. In one embodiment of the invention, theintravenous formulation comprises the compound of formula S-(1) given ata high dose.

In another embodiment of the invention, an additional drug in theregimen comprises an anti-mitotic agent or anti-microtubule agent. Inone variation, the dose of a single infusion of the compound of formulaS-(1) exceeds 160 mg/m². In another variation, the dose of a singleinfusion of the compound of formula S-(1) exceeds 185 mg/m².

The application also provides compositions for and a method to produceprolonged elevated plasma levels of the compound of formula S-(1) topromote synergistic interaction between the compound of formula S-(1)and a one or more other chemotherapeutic agents, wherein: the compoundof formula S-(1) is administered as an intravenous formulation, and thecompound of formula S-(1) is administered on the day of, or within 3days of administration of the one or more other chemotherapeutic agents.In one aspect of the invention, the one or more other chemotherapeuticagents comprises an anti-mitotic agent or anti-microtubule agent. Inanother aspect of the invention, the intravenous formulation comprisestemozolomide and/or bevacizumab (Avastin™).

The application also provides compositions for and a method of bindingmicrotubules in cancer cells comprising contacting the cells with aneffective amount of the compound of formula S-(1). The application alsoprovides compositions for and a method of hindering or preventingmitosis in cancer cells comprising contacting the cells with aneffective amount of the compound of formula S-(1). In one aspect of theinvention, the cancer cells are pancreatic, breast, or colorectal cancercells.

The application also provides compositions for and a method of assayingfor cancer cell sensitivity to the compound of formula S-(1) comprising:a) providing a cancer cell; b) contacting the cancer cell with acomposition comprising the compound of formula S-(1); c) analyzing thecancer cell for inhibition of growth; and d) comparing the inhibition ofgrowth in the cancer cell from step (c) with the inhibition of growth inthe cancer cell in the absence of the compound of formula S-(1), whereingrowth inhibition by the compound of formula S-(1) indicates that thecancer cell is susceptible to the compound of formula S-(1).

In one embodiment of the invention, the method further comprisesassaying for mitotic arrest in a cancer cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. S-(1) crosses rat blood-brain barrier.

FIG. 2. Mouse Plasma and Brain Levels of S-(1) after single IV dose.

FIG. 3. Summary of U251 Orthotopic Intracranial Xenograft Study Data.

FIGS. 4A, 4B, and 4C. S-(1) Oral efficacy in Mice.

FIGS. 5A and 5B. Oral Efficacy of S-(1) in Neuroblastoma Xenograft.

FIGS. 6A and 6B. S-(1) Oral Efficacy in Ovarian Tumor Xenograft.

FIGS. 7A and 7B. S-(1) Oral efficacy in Glioblastoma Xenograft.

FIGS. 8A and 8B. IV efficacy of S-(1) in Glioblastoma Xenograft Model.

FIGS. 9A and 9B. Efficacy Comparison in Prostate Tumor Xenograft Model.

FIGS. 10A and 10B. Efficacy Comparison in Lung Tumor Xenograft Model

FIGS. 11A and 11B. Efficacy Comparison in Breast Tumor Xenograft Model

FIGS. 12A and 12B. Efficacy Comparison in HT 29 Colon Xenograft

FIGS. 13A and 13B. Oral S-(1)+/−Avastin

FIGS. 14A and 14B. Combination Efficacy of S-(1)+/−Avastin

The compound of formula S-(1) may be administered by any conventionalroute of administration including, but not limited to, oral, pulmonary,intraperitoneal (ip), intravenous (iv), intramuscular (im), subcutaneous(sc), transdermal, buccal, nasal, sublingual, ocular, rectal andvaginal. It will be readily apparent to those skilled in the art thatany dosage or frequency of administration that provides the desiredtherapeutic effect is suitable for use in the present application.

The therapeutically effective amount of the compound of formula S-(1)may be administered by week or by 21 days for a total dose of about7-555 mg/m², or 7-240 mg/m², or 7-185 mg/m² during a 21 day period. Thedosages, however, may be varied depending on the requirements of thesubject to be treated, including sex, age, weight, diet, etc. Theprecise amount of the compound of formula S-(1) required to beadministered depends on the judgment of the practitioner and is peculiarto each individual.

Another embodiment of the present application is a pharmaceuticalcomposition for treating brain tumor, comprising a therapeuticallyeffective amount of the compound of formula S-(1) and a pharmaceuticallyacceptable carrier.

Another embodiment of the present application is a pharmaceuticalcomposition for oral administration of the compound being patientfriendly. The solubility properties of the compound of formula S-(1) aresuch that high concentration of the compound of formula S-(1) can beachieved in pharmaceutically acceptable carriers, between 75 and 200mg/ml, that can be filled in hard and soft gelatin capsules with anappropriate chemical and physical stability profile.

To prepare a pharmaceutical composition of the present application, thecompound of formula S-(1) is admixed with a pharmaceutically acceptablecarrier according to conventional pharmaceutical compounding techniques,wherein the carrier may take a wide variety of forms depending on theform of preparation desired for administration. Suitablepharmaceutically acceptable carriers are well known in the art.Descriptions of some pharmaceutically acceptable carriers may be foundin The Handbook of Pharmaceutical Excipients Eds. Rowe et al., AmericanPharmaceutical Association and the Pharmaceutical Society of GreatBritain.

The pharmaceutical composition of the present application may be,depending on the route of administration, in the form of a tablet, pill,capsule, granule, powder, ointment, gel, solution, sterile parenteralsolution or suspension, metered aerosol or liquid spray, or suppositorythat may contain micro or nano particles, a colloid, liposome or abiocompatible or biodegradable medium.

As a solid dosage form, the pharmaceutical composition of the presentapplication may comprise, in addition to the compound of formula S-(1),at least one diluent, binder, adhesive, disintegrant, lubricant,antiadherent, and/or glidant. Additionally, sweeteners, flavorants,colorants and/or coatings may be added for specific purposes.

As a liquid dosage form, the pharmaceutical composition of the presentapplication may comprise, in addition to the compound of formula S-(1)and a liquid vehicle, at least one wetting agent, dispersant,flocculation agent, thickener, buffer, emulsifier, amphiphilic, osmoticagent, coloring agent, anti-oxidant, flavor, fragrance, and/orpreservative.

Such a possible liquid formulation includes a sterile solutioncontaining 10 mg/mL of the compound of formula S-(1) in a 15:85 or 50:50(w/v) polyoxyl 35 castor oil/dehydrated alcohol solution. An appropriatepharmaceutical grade polyoxyl 35 castor oil is Cremophor EL-P, which isa non-ionic solubilizer made by reacting castor oil with ethylene oxidein a molar ratio of 1:35, followed by a purification process (BASFPharma).

Other suitable agents for inclusion in such formulations are asdescribed in WO 99/45918. Such additional carriers include vitamin ETPGS, oleic acid, Tweens such as Tween 80, lipid carriers used forparenteral nutrition, solutrol and the like. As shown in Table 10 below,these agents and the composition of the final formulation might have animpact of the pharmacokinetics of the compound and its pharmacodymanicsand thus its tissue distribution.

As previously mentioned, the compound of the invention surprisingly maybe administered orally as further exemplified below. Oral administrationmay involve swallowing, so that the compound enters the gastrointestinaltract, or buccal or sublingual administration may be employed by whichthe compound enters the blood stream directly from the mouth.

The therapeutically effective amount of the compound of formula S-(1)may be administered by the parenteral route using a weekly or 21 daysschedule for a total dose of about 7-555 mg/m², or 7-240 mg/m², or 7-185mg/m² during a 21 day period. The dosages, however, may be varieddepending on the requirements of the subject to be treated, includingsex, age, weight, diet, etc. The precise amount of the compound offormula S-(1) required to be administered depends on the judgment of thepractitioner and is peculiar to each individual.

The compound of formula S-(1) may be administered by the oral routeusing a daily, weekly or 21 days schedule, and any schedule comprisedwithin this range, for a total dose of about 5-600 mg/m², or 25-400mg/m². The dosages, however, may be varied depending on the requirementsof the subject to be treated, including sex, age, weight, diet, etc. Theprecise amount of the compound of formula S-(1) required to beadministered depends on the judgment of the practitioner and is peculiarto each individual.

It is believed that the compounds and hence compositions of thisinvention possess an enhanced safety profile (for example, on the immunesystem or the blood) in comparison to marketed taxanes which offers thepotential for enhanced or longer dosing schedules under the direction ofthe skilled physician than marketed taxanes.

The compositions may for oral administration contain at least 15 mg ofthe compound of the invention per dose, more aptly at least 50, 80, 100,150, 200 mg and more aptly less than 250 mg per dose. However, thedosage may be varied as directed by the physician in view of theindividual patient's response. A liquid composition will normallycontain about 0.1 mg/ml to about 300 mg/ml for example about, 1, 5 10,50, 100 or 150 mg/ml of the compound of the invention. A non-liquidcomposition may contain a proportion of the compound of the invention,for example 5% to 50%, such as 10, 20, 25 or 30% by weight. In the caseof a liquid composition for parenteral administration, the compositionwill normally contain between 1 mg/ml and 50 mg/ml while the compositionwill normally contain between 40 mg/ml to 200 mg/ml if administeredorally. WO 1999/45918 and the international patent applications and USpatent applications referred to above discloses compositions that may beconsidered for use with compounds of the invention.

Formulations suitable for oral administration include solid formulationssuch as tablets, capsules containing particulates, liquids, or powders,lozenges (including liquid-filled), chews, multi- and nano-particulates,gels, solid solution, liposome, films, ovules, sprays and liquidformulations. Liquid formulations include suspensions, solutions, syrupsand elixirs. Such formulations may be employed as fillers in soft orhard capsules and typically comprise a carrier, for example, ethanol,polyethylene glycol, propylene glycol, methylcellulose, or a suitableoil, a lipohilic component with a high HLB value, an amphiphilicsolvent, and one or more emulsifying agents and/or suspending agents.Liquid formulations may also be prepared by the reconstitution of asolid, for example, from a sachet. The compound of the invention mayalso be used in fast-dissolving, fast-disintegrating dosage forms.Tablets generally contain a disintegrant. Examples of disintegrantsinclude sodium starch glycolate, sodium carboxymethyl cellulose, calciumcarboxymethyl cellulose, croscarmellose sodium, crospovidone,polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose,lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinisedstarch and sodium alginate.

Binders are generally used to impart cohesive qualities to a tabletformulation. Suitable binders include microcrystalline cellulose,gelatin, sugars, polyethylene glycol, natural and synthetic gums,polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose andhydroxypropyl methylcellulose. Tablets may also contain diluents, suchas lactose (monohydrate, spray-dried monohydrate, anhydrous and thelike), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystallinecellulose, starch and dibasic calcium phosphate dihydrate.

Tablets may also optionally comprise surface active agents, such assodium lauryl sulfate and polysorbate 80, and glidants such as silicondioxide and talc. Tablets may also generally contain lubricants such asmagnesium stearate, calcium stearate, zinc stearate, sodium stearylfumarate, and mixtures of magnesium stearate with sodium laurylsulphate. Other possible ingredients include anti-oxidants, colourants,flavouring agents, preservatives and taste-masking agents. Tablet blendsmay be compressed directly or by roller to form tablets. Tablet blendsor portions of blends may alternatively be wet-, dry-, ormelt-granulated, melt congealed, or extruded before tabletting. Thefinal formulation may comprise one or more layers and may be coated oruncoated; it may even be encapsulated.

Consumable oral films are typically pliable water-soluble orwater-swellable thin film dosage forms which may be rapidly dissolvingor mucoadhesive and typically comprise the compound of formula S-(1), afilm-forming polymer, a binder, a solvent, a humectant, a plasticiser, astabiliser or emulsifier, a viscosity-modifying agent and a solvent.Some components of the formulation may perform more than one function.The film-forming polymer may be selected from natural polysaccharides,proteins, or synthetic hydrocolloids and is typically present in therange 0.01 to 99 weight %, more typically in the range 30 to 80 weight%.

Other possible ingredients include anti-oxidants, colorants, flavouringsand flavour enhancers, preservatives, emulsifiers, salivary stimulatingagents, cooling agents, co-solvents (including oils), emollients,bulking agents, anti-foaming agents, surfactants and taste-maskingagents.

Solid formulations for oral administration may be formulated to beimmediate and/or modified release. Modified release formulations includedelayed-, sustained-, pulsed-, controlled-, targeted and programmedrelease.

Biological Examples

In Vitro ED₅₀ MT Polymerization Study

In this tubulin binding assay, microtubule protein (MTP) is used as asubstrate. The assay contains bovine tubulin plus microtubule associatedproteins (MAP). MTP is polymerized into microtubules in the presence ofDAPI (4′,6′-diamidino-2-phenylindole), a fluorescent compound. DAPIbinds to tubulin; when microtubules are formed and there is anenhancement of fluorescence. The microtubule formation is measured as afunction of time, using a fluorescence plate reader. The ED₅₀ valuesobtained with this method are in good agreement with older sedimentationtechniques. The more current assay, using DAPI, is faster and uses lessprotein. The method used is based on the procedure published by Donna M.Barron, et al, “Fluorescence-based high-throughput assay forantimicrotuble drugs” Analytical Biochemistry, 315: 49-56, 2003, whichis incorporated by reference in its entirety. The excitation wavelength,in that assay, was set at 370 nm and the emission wavelength was set at450 nm for the DAPI experiments.

A Bio-Tek FL 600 microplate Fluorescence Reader was used to measure therelative level of fluorescence in the DAPI assay.

Assays were conducted in 96-well plates. Each well contained a totalvolume of 0.1 mL consisting of PEM buffer (0.1 M Pipes, 1 mM EGTA, 1 mMMgCl₂, pH 6.9), 0.2 mg bovine microtubule protein, and 10 μg of DAPI.Compounds having paclitaxel-like activity of varying concentrationsdissolved in DMSO were added last. The final DMSO concentration was 4%.The plates were incubated at 37° C. for 30 minutes and read in afluorescence plate reader using an excitation wavelength of 360 nm andan emission wavelength of 460 nm. Fluorescence values were corrected forthe sample without compound. Results were expressed as a percent ofmaximum assembly, with maximum assembly taken to be that obtained at 25μM paclitaxel.

Experiments were done twice in triplicate. Results were subsequentlycombined and fit to a non-linear regression program. The results fromthese studies summarized in Table 2 indicate that the S-(1)diastereoisomer has an ED₅₀ potency that is equal to or greater thanthat determined for other tubulin binding agents such as paclitaxel,docetaxel and Epothilone B.

TABLE 2 Summary of Tubulin Polymerization Assays, Comparison of S-(1) topaclitaxel, docetaxel, and epothilone B. Compound ED50, μM ED50Compound/ED50 Paclitaxel S-(1) 1.58 ± 0.46 0.53 Paclitaxel 2.97 ± 0.501.00 Docetaxel 3.18 ± 0.45 1.07 Epothilone B 3.31 ± 0.51 1.11

MTS Proliferation Assay (Promega)

Day 1: Cells were plated in appropriate growth medium at 5×10³ per wellin 100 ul in 96 well tissue culture plates, Falcon, one for each drug tobe tested. Col 1 was blank; it contained no cells, just medium. Theplates were incubated overnight at 37° C., 5% CO₂ to allow attachment.

Day 2: Added 120 ul growth medium in wells of 96-well “dilution plates”(one for each drug) and let sit in 37° C. incubator for about 1 hr.

Thawed DMSO drug stocks (usually at 10 mM). Each drug was diluted 6 ulinto a tube with 3 ml growth medium, to 20 μM.

Aspirated medium from col 12 of a dilution plate; added 200-300 ul of 20uM drug to wells of col 12. Made serial dilution down this 96-wellplate: for a 1:5 dilution pattern, moved 60 ul from col 12 to col 11,mixed 4-5 times (using 8 place multi-pipettor), moved 60 ul to col 10,etc. stopping at col 3.

Moved 100 ul of medium+drug from dilution plate to a cell plate, i.e.col 1 from drug plate (blank=no cells) to col 1 of cell plate, etc. upto col 12. Col 2 contained cells with no drug. Col 3 had the lowestconcentration of drug (0.005 nM) and col 12 had the highest drugconcentration (100 μM).

Day 4 or 5: Terminated the assay 48 to 72 hrs after drug addition.Thawed MTS reagent; made up enough medium+MTS to cover all plates at 115ul per well (100 ul medium+15 ul MTS). Aspirated medium+drugs from cellplate; replaced with medium+MTS mix and incubated 1-6 hrs (37° C., 5%CO₂), depending on cell type. When the color turned dark in controlwells (col 2), and was still light in col 12, the absorbance at 490 nmwas read on a plate reader; the results were used to calculate IC₅₀.

The effects of the compound of formula S-(1) measured in in vitro andxenograft animal models of various brain cancers were evaluated in thefollowing experimental examples, which are intended to be a way ofillustrating but not limiting the present application.

S-(1) Pharmacokinetic Data in Mouse Model:

S-(1) was formulated in 7% Ethanol: 3% Cremophor EL: 90% D5W (5%Dextrose in water). The formulated drug was administered as a single IVbolus via tail vein injection. The details are set out below:

Animal Information:

Total No. Food Species Strain Sex Wt. Range (g) Required Source FastedReturned Mice CD-1 Male 20-30 36 CRL No NAStudy Design

Dose Group No. of Test Volume Conc. Dose No. Animals Article VehicleRoute (mL/kg) (mg/mL) (mg/kg) 1 36 S-(1) 7% Ethanol: IV 10 2 20 3%Cremophor (Tail EL: 90% D5W Vein)

Sample Collection timing: Terminal blood (plasma) and brain specimenswere collected at 9 times post-dose, with 4 mice per sample time. Samplecollection times: 5, 15 min., 2, 8, 24, 32, 48, 72 and 96 hr post-dose(4 mice per sample time).

Blood collection: A terminal blood sample was collected from each mousevia cardiac puncture. The whole blood was collected on wet ice, spundown immediately (at 4° C.), and plasma stored frozen at −20° C. untilanalysis. The anticoagulant used was K₂EDTA. Brain Sample Collection:Whole brains were removed by dissection. Brains were collected on wetice, weighed, and stored at −20° C.

TABLE 3 Mouse Plasma and Brain Levels of S-(1) after Single iv DoseBrain Plasma Cmax ng/ml Cmax ng/ml  3726.6  1550.2 AUC(0-t) ng-hr/ml AUC(0-t) ng-hr/ml 64984.9 16870.2 AUC (0-∞) ng-hr/ml AUC (0-∞) ng-hr/ml88742.9 16951.2 MRT (expo)hr MRT (expo)hr   42.5   17.6 CL ml/hr CLml/hr   0.006   0.024

The results indicate that S-(1) is not impeded by the blood-brainbarrier in the mouse model. See FIG. 2.

Antitumor Efficacy of S-(1) Against Human U251 CNS Tumor Cells Implantedin Mouse Brain:

Additional studies were conducted in animal models to determine theefficacy of S-(1) against brain tumors. In one study, the anti-tumoractivity of S-(1) was evaluated both when administered alone and incombination with temozolomide, against intracerebrally (ic) implantedhuman U251 (glioblastoma) CNS tumor cells in male athymic nude mice.

Tumor Model: Each animal was implanted with one million U251 human CNStumor cells from an in vitro cell line by ic injection with a 25 gaugeneedle. The day of tumor implantation (May 24, 2006) was designated asday 0. A sufficient number of mice were implanted so that animals withbody weights in a range as narrow as possible were selected for thetrial on the day of treatment initiation (day 1 after tumorimplantation). Animals were randomly assigned to the treatment groupsand individually identified by earmark codes.

Drug Formulation: On each day of treatment, the appropriate amount ofS-(1) was formulated in 3% cremophor EL/7% ethanol/90% D5W at aconcentration of 2 mg/mL. A portion of this solution was then dilutedwith the complete vehicle to achieve the lower dosing concentration of1.2 mg/mL. Both concentrations of S-(1) were then kept at 37° C. andinjected within 30 minutes of formulation on the basis of exact bodyweight using a volume of 0.1 mL/10 g of body weight. Temozolomide(Temodar, Schering Corporation) was prepared on each day of injection inKlucel+tween 80 at a concentration of 4 mg/mL. Temozolomide wasadministered within 5 minutes of formulation on the basis of exact bodyweight using a volume of 0.2 mL/10 g of body weight.

Data Collection: Animal health surveillance was conducted and mortalitydata were collected daily. The animals were weighed twice weeklystarting with the first day of treatment.

Study Duration: The study was terminated 100 days after tumorimplantation. Any animal found moribund or whose body weight droppedbelow 14 g was euthanized prior to study termination.

Parameters Evaluated: Number of 100-day survivors, median day of death,and the increase in lifespan based on median day of death and expressedas a percentage (% ILS), median survival time and the % ILS based onmedian survival time.

Results:

The vehicle-treated control group had a median day of death and a mediansurvival time of 10 days. All animals died or were euthanized due tomoribundity between days 9 and 11. The maximum loss in mean body eightwas 32% (7 g).

Due to the dehydrating effect of treatment with S-(1), animals in groups2, 3, 5, and 6 that received treatment with S-(1) at a dosage of 20 or12 mg/kg/dose were given 5% dextrose in lactated Ringer's solution tolessen the debilitating effects of the treatment. Administration of theRinger's solution was initiated when animal body weights dropped bygreater than 10% and continued until the animal recovered, died, or waseuthanized.

Intravenous treatment with S-(1), administered at dosages of 20 and 12mg/kg/dose on a q4d×3 schedule, resulted in a 60% and 45% ILS,respectively, whether the calculation was based on day of death orsurvival time. The corresponding median days of death and the mediansurvival times were 16 and 14.5 days for the S-(1) dosages of 20 and 12mg/kg/dose, respectively. One death occurred on day 2 in the groupreceiving treatment with the dosage of 20 mg/kg/dose. This death mayhave been treatment-related or may have been a delayed effect of theanesthesia used at the time of the ic tumor implant.

Treatment with temozolomide administered at a dosage of 80 mg/kg/dosegiven po q4d×3 was quite effective against the growth of the U251 CNStumor cells with an ILS of 380% when calculated based on median day ofdeath (48 days) and 400% when calculated based on median survival time(50 days). There was one survivor in this group at the time of studytermination on day 100. Treatment with temozolomide was toleratedwithout treatment-related deaths and with only a minimal loss (8%, 2 g)in mean body weight.

Administration of S-(1) given iv at a dosage of 20 mg/kg/dose incombination with temozolomide given po at a dosage of 80 mg/kg/doseresulted in an ILS of 575%, with the median day of death and the mediansurvival time both being 67.5 days. The maximum loss in mean body weightloss observed in this treatment group was 22% (5 g). The loss wasrecovered following cessation of treatment. One death occurred on day 5,two deaths occurred on day 9, and one death occurred on day 16, possiblyfrom toxicity of S-(1). The remaining six animals in the group didrespond to therapy with deaths occurring between days 64 and 90.

The group receiving treatment with S-(1) at a dosage of 12 mg/kg/dose incombination with temozolomide at 80 mg/kg/dose responded more favorablythan the group receiving the higher dosage of S-(1). The median day ofdeath was day 73, with an ILS of 630%, and the median survival time was75 days with an ILS of 650%. Results are shown in FIG. 3.

TABLE 4 Summary of U251 Orthotopic Intracranial Xenograft Study S-(1) 20mg/kg, ip. 60% ILS (survivors day QD1, 5, 9 100 = 0) Group 2 Group 2 vs.2, P = 0.000 S-(1) 12 mg/kg, ip. 45% ILS (survivors day QD1, 5, 9 100 =0) Group 3 Group 1 vs. 3 P = 0.000 TMZ 80 mg/kg, po. 380% ILS (survivorsday QD1, 5, 9 100 = 1) Group 4 Group 2 vs 3, P = 0.450 TMZ 80 mg/kg,po. + S-(1) 575% ILS (survivors day 20 mg/ip. 100 = 1 QD1, 5, 9 Group 4vs 5, P = 0.683 Group 5 TMZ 80 mg/kg, po. + S-(1) 650% ILS (survivorsday 12 mg/kg ip. 100 = 3) QD1, 5, 9 Group 4 vs. 6, P = 0.046 Group 6Group 5 vs. 6, P = 0.108

Monotherapy with temozolomide produced an excellent effect against theU251 human CNS tumor cells, while S-(1), administered alone, produced amoderate effect. Combination treatment with S-(1) and temozolomide wasquite effective at both dosages of S-(1). The better response was seenin the combination group receiving the lower S-(1) dosage, possiblybecause of toxicity of S-(1) at the dosage of 20 mg/kg/dose.

Toxicology. Central Nervous System (CNS) safety study in rats:

The objective of this study was to evaluate the acute pharmacologicaleffects of S-(1) on the central nervous system following intravenousadministration in the male albino rat. This study was conducted atClinTrials BioResearch (CTBR, Montreal, Quebec, Canada) as a GLP study.

Results:

All animals were observed twice daily for signs of ill health orreaction to treatment, except on day of arrival and necropsy. AFunctional Observation Battery (FOB) assessment, along with gripstrength, hind limb splay and body temperature measurements wereperformed for all animals once pre-dose and at approximately 15 minutes,1 hour, 4 hours, 8 hours and 24 hours post-dose. After the lastobservation, all animals were euthanized without further examination.There were no deaths or treatment-related clinical signs.

A single intravenous administration of S-(1) at 6.25, 12.5 or 25 mg/kghad no biological effect on central nervous system, when measured byqualitative assessment of the functional observational battery andquantitative assessment of grip strength, hind limb splay, and bodytemperature at approximately 15 minutes, 1, 4, 8 and 24 hours post-dose.

TABLE 5 Results of CNS Safety Study Dose Dose No. of Group/ Dose LevelConcentration Volume Animals Identification (mg/kg) (mg/kg) (mL/kg)Males 1. Control 0 0 3 8 2. S-(1) 6.25 2.08 3 8 3. S-(1) 12.5 4.17 3 84. S-(1) 25 8.33 3 8 Conclusions: Dose levels of 6.25, 12.5 and 25 mg/kghad no biological effects on the central nervous system of male SpragueDawley rats following administration by intravenous injection. The noadverse effect level for this study is 25 mg/kg.

Clinical Studies of S-(1)

Two Phase 1 trials examining the safety and pharmacokinetics of multipleascending doses of single agent, intravenous S-(1), when administered intwo different treatment schedules, are ongoing. Both trials haveenrolled patients with advanced solid tumors, non-Hodgkin's lymphoma orHodgkin's lymphoma that have recurred or progressed following at leaststandard therapy; patients with malignancies for which there is nostandard therapy; patients who are not candidates for standard therapy;or patients who have chosen not to pursue standard therapy. In StudyS-(1)-01, intravenous S-(1) is administered weekly for 3 consecutiveweeks followed by one week of no therapy; treatment cycles are repeatedevery 28 days in patients who remain eligible for continued treatment.Study S-(1)-01 is currently being conducted in the United States. InStudy S-(1)-02, intravenous S-(1) is administered every 3 weeks;treatment cycles are repeated every 21 days in patients who remaineligible for continued treatment. Study S-(1)-02 is currently beingconducted in the United States and in Israel.

TABLE 6 Clinical Studies of S-(1) Study No. Patients Enrolled Phase DoseGroup Protocol Regimen S-(1)-01 27 S-(1) i.v. in a 50% cremaphor, 50%Phase 1    7 mg/m²: 4 pts ethanol formulation over 1 hour or less   14mg/m²: 4 pts weekly × 3 followed by 1 week of no   28 mg/m²: 4 ptstherapy (28-day treatment cycle)   56 mg/m²: 4 pts 127.5 mg/m²: 7 pts  185 mg/m²: 4 pts S-(1)-02 20 S-(1) i.v. in a 15% cremphor, 85% Phase 1  56 mg/m²: 2 pts ethanol formulation over 1 hour or less   84 mg/m²: 1pt every 3 weeks (21-day treatment cycle)   126 mg/m²: 3 pts   185mg/m²: 6 pts   160 mg/m²: 8 ptsClinical Safety—Adverse Events:

Dose Limiting Toxicity: Dose limiting toxicity has been observed in onepatient in Study S-(1)-01 who received 185 mg/m² of S-(1). The doselimiting toxicity was Grade 3 sensory neuropathy. Dose limiting toxicityhas been observed in 2 patients in Study S-(1)-02 at a dose of 185mg/m². In both patients, the dose limiting toxicity was Grade 3 sensoryneuropathy.

Clinical Pharmacokinetics:

Pharmacokinetic data for the Phase 1 studies S-(1)-01 and S-(1)-02 aresummarized in Table 7. Plasma pharmacokinetics of S-(1) were assessedfollowing the first dose of S-(1) in both studies. Plasma samples wereanalyzed for S-(1) using a validated LC/MS-MS method. Two differentformulations of S-(1) were administered in the two different Phase 1studies: a 50:50 (w/v) formulation of Cremophor EL-P/ethanol,administered in the S-(1)-01 study, and a 15:85 (w/v) formulation ofCremophor EL-P/ethanol, administered in the S-(1)-02 study.

Analysis of the PK data revealed that the plasma AUC of S-(1) appearedto be relatively dose proportional, with a moderate level ofinterpatient variability. Plasma t_(1/2) values ranged from 3.45-8.4hours for the S-(1)-01 study, and from 3.43-8.96 for the S-(1)-02 study.Clearance did not appear to be dose-dependent in either study. There didnot appear to be any difference in pharmacokinetic parameters, includingV_(ss), for the two different formulations of S-(1).

TABLE 7 Plasma Pharmacokinetic Parameters for S-(1) Dose AUC₀₋₂₄ Cl Vsst_(1/2) (mg/m²) (ng/ml * hr) (L/hr/m²) (L/m²) (hr) Study S-(1)-01 (50:50Cremophor EL-P/ethanol formulation) 14 457 ± 297 35.5 ± 29.7 23.2 ± 74.5 8.4 ± 11.5 28 682 ± 322 43.5 ± 14.5 309 ± 161 3.45 ± 1.09 56 2070 ±888  28.6 ± 13.2 216 ± 70  5.66 ± 3.63 85 5970 ± 3900 16.6 ± 9.26  142 ±75.6 5.52 ± 5.26 127.5 4460 ± 2140 30.2 ± 11.9 239 ± 116 6.59 ± 2.60Study S-(1)-02 (15:85 Cremophor EL-P/ethanol formulation) 56 2530 ± 847 18.9 ± 9.53  232 ± 93.6 8.96 ± 7.09 84 3710 15.4 300 — 126 6110 ± 303022.1 ± 11.2 166 ± 105 6.27 ± 3.64 185 7260 ± 2620 24.7 ± 12.2 215 ± 1163.43 ± 7.31Summary of a Study of S-(1): Administered Weekly in Patients withAdvanced Cancer:

In preclinical studies, S-(1) demonstrated antitumor activity againstmultiple human tumor xenografts in nude mice, including xenografts thatexpressed mdr-1 and that were resistant to other taxanes. The safety andtolerability of S-(1) when administered weekly for 60 minutes for 3weeks followed by a 1 week rest (4 week cycle) was examined in thisPhase 1 dose escalation study in patients (pts) with advanced neoplasms.

Treatment cohorts consisted of 3 pts and were expanded to 6 pts in theface of dose-limiting toxicity (DLT); pts could remain on study untilthe development of progressive disease or an intolerable adverse event.DLT was defined as Gr 4 heme toxicity lasting 7 days; febrileneutropenia, Gr 3 thrombocytopenia with bleeding, Gr 3 elevation oftransaminases lasting 7 days or any other Gr 3/4 toxicity other thannausea or vomiting.

Results: 25 pts (M:F 16:9, median age 60, range 24-86) were enrolled in7 dose levels ranging from 7-185 mg/m². Pts' cancers included colorectal(6 pts); NSCLC (2); prostate (2); squamous cell carcinoma (2) and 1 μLeach with cervical, breast, ovarian, gastric, pancreatic, bladderendometrial, NSCLC, SCLC, glioblastoma, melanoma, renal cell andhepatocellular carcinoma. All pts but 1 had received prior chemotherapy(median no. prior treatments: 3 (range, 1-7). Drug related adverseevents included nausea, vomiting, diarrhea, fatigue, anorexia, rash,anemia and peripheral neuropathy. DLT of Grade 3 peripheral neuropathyhas been observed. PK data to date reveal that AUC is generally doselinear. At a dose of 127.5 mg/m², clearance was 30.2±11.9 L/hr/m² andt_(1/2) 8.6±1.3 hrs. Antineoplastic activity was seen in a patient withpancreatic cancer.

In conclusion, S-(1) can be safely administered in doses of up to 185mg/m² weekly×3 in heavily pre-treated patients. It is predicted that inchemo-naive patients, or patients with more limited exposure tochemotherapy, that the dose could be escalated even further. S-(1) hasactivity in pancreatic cancer. PK is dose linear and predictable.

Summary of a Phase 1 Study of S-(1): Administered Every 21 Days inPatients with Advanced Cancer:

In preclinical studies, S-(1) suppressed the growth of multiple humantumor xenografts in nude mice, including xenografts that expressed mdr-1and that were resistant to other taxanes. The safety and tolerability ofS-(1) when administered every 21 days was examined in this Phase 1 doseescalation study in patients (pts) with advanced neoplasms.

S-(1) was administered over 1 hour every 21 days in ascending doses togroups of 3 pts. Treatment cohorts were expanded to 6 pts in the face ofdose-limiting toxicity (DLT); pts could remain on study until thedevelopment of progressive disease or an intolerable adverse event. DLTwas defined as Gr 4 heme toxicity lasting 7 days; febrile neutropenia,Gr 3 thrombocytopenia with bleeding, Gr 3 elevation of transaminaseslasting 7 days or any other Gr 3/4 toxicity other than nausea orvomiting.

Results: 14 patients (M:F 5:9, median age 58.5, range 49-77) wereenrolled in 5 dose levels ranging from 56-185 mg/m². Patients' cancersincluded colorectal (5 pts), esophageal (2), pancreatic (2), NSCLC (2),breast (2) and ovarian (1). All patients had received prior chemotherapy(median no. prior treatments: 3 (range, 2-10)). Drug related adverseevents included mucositis, vomiting, diarrhea, neutropenia,thrombocytopenia, myalgias and peripheral neuropathy. Only 1 μL.experienced Gr 4 neutropenia. DLT of Gr 3 peripheral sensory neuropathywas observed at a dose of 185 mg/m². At a dose of 160 mg/m² no DLT wasobserved. 1 μL with pancreatic cancer had a confirmed response to S-(1).PK data reveal that AUC is generally dose linear. At a dose of 126mg/m², clearance was 24.7±12.2 L/hr/m² and t_(1/2) was 10.6±7.1 hrs.

It was determined that S-(1) can be safely administered in a dose of 160mg/m² every 21 days in heavily pre-treated patients. It is predictedthat in chemo-naive patients, that the dose could be escalated evenfurther. The dose limiting toxicity was Gr 3 peripheral neuropathy.S-(1) appears to have activity in pancreatic cancer. PK is dose linearand predictable.

Oral Dosing of the Compound of Formula S-(1):

The compound of formula S-(1) when dosed either oral or iv showsefficacy in mouse xenografts. From these efficacy studies and MTDstudies, the “apparent oral bioavailability” of the compound of formulaS-(1) in nude mice would be in the 40 to 80% range.

TABLE 8 Formula S-(1) Injection Formulation Administered Orally: MousePK Parameters S-(1) - IV 10/mg/kg S-(1) - Oral 10 mg/kg C_(max) (nM) naC_(max) (nM) 460 t_(max) (hr) na t_(max) (hr) 6.0 t½ (hr) 6.1 t½ (hr)5.1 AUC_(last) (nM · hr) 6298 AUC_(last) (nM · hr) 3434 AUC_(inf) (nM ·hr) 7738 AUC_(inf) (nM · hr) 3609 CL (L/hr/kg) 1.49 F (%) 47 Vdss (L/kg)13.35

Efficacy of Orally Dosed S-(1) in Mouse Neuroblastoma Xenografts:

Summary of Experimental Protocol: Human neuroblastoma tumor cells wereimplanted via subcutaneous injection of 1-10×10⁶ cells in nude mice.Tumors were allowed to grow to 200 mg+/−50 mg in size. Drug dosing wasinitiated, and tumor volume and body weight were recorded twice weekly.

Data Analysis:

The mean value for % body weight change and % tumor volume increase foreach experimental group was plotted including error bars for thestandard error of the mean. Other metrics for assessing antitumoreffects include: % T/C values as calculated by the following formula:

$\frac{\%\mspace{14mu}{Mean}\mspace{14mu}{Tumor}\mspace{14mu}{Volume}\mspace{14mu}{of}\mspace{14mu}{Treated}\mspace{14mu}{Group}}{\%\mspace{14mu}{Mean}\mspace{14mu}{Tumor}\mspace{14mu}{Volume}\mspace{14mu}{of}\mspace{14mu}{Control}\mspace{14mu}{Group}}$Log  Kill = (T − C/3.32  ×  (T d)

-   -   T is the time in days for the median tumor volume to reach 1        gram in treated group    -   C is the time in days for the median tumor volume to reach 1        gram in control group    -   Td is the tumor doubling time in days    -   Cures are excluded from T-C calculations

Results are shown in FIGS. 4 to 9.

S-(1) Rat Oral Bioavailability:

IV formulation (Cremophor El P/alcohol) and a suspension of the compoundof formula S-(1) in aqueous medium.

Several studies were performed to investigate specific parameters. Itwas found that there is a gender difference but it does not influencebioavailability (F) overall, the formulation dilution does not impact F,there is saturation of the absorption that seems formulation dependent,and the data is reproducible between experiments.

From the solubility and compatibility studies between the compound offormula S-(1) and excipients that could be used, a reasonable list offormulations were evaluated in vivo. Pharmacokinetics results for 9formulations administered to female rats by oral gavage are listed inTable 9 below.

TABLE 9 AUC of Selected Evaluated Oral Formulations AUC last AUCinf/Dose(hr * ng/ml) (hr * kg * ng/mL/mg) Cremophor:EtOH 50:50/dil. 1:9 2220 426Tween 80:EtOH:Labrafil:Labrasol 25:25:35:35/dil. 1:1 5740 427Solutol:EtOH 60:40/dil. 1:1 6590 503 Vit E-TPGS:Cre:EtOH:D5W50:17:17:16/dil. 1:3 1450 266 Solutol:Cre:EtOH:D5W 50:17:17:16/dil. 1:32370 431 Lutrol:Cre:EtOH:D5W 40:20:20:20/dil. 1:3 2380 436 OleicAcid:Cre:Captex:Capmul:D5W 8:25:33:17:17/dil. 2610 555 1:3Solutol:EtOH:Labrafil:labrasol 50:10:25:15/dil. 1:1 2850 475Gelucire:Labrafil:PEG300 60:20:20/dil. 1:1 3120 520

In addition, when contemplating oral administration, one typically triesto evaluate the oral bioavailability of the compound. Using the 50:50Cremophor:Ethanol formulation as a case study given orally (AUC last:2540; norm AUCinf: 477), F was calculated using two different IVformulations as reference, the 50:50 Cremophor:Ethanol and the 15:85Cremophor:Ethanol As shown in Table 10 below, the calculated F value issignificantly impacted by the PK values of the IV reference.

TABLE 10 Calculated F AUC last AUCinf/Dose Calculated F (hr * ng/ml)(hr * kg * ng/mL/mg) (%) Cremophor:EtoH 14600 2040 ~23% 50:50/dil. 1:9dosed IV Cremophor:EtoH 4470 970 ~49% 15:85/dil. 1:9 dosed IVAccordingly, the invention comprises a polyethoxylated castor oilformulation comprising the compound of formula S-(1). Preferably, thepolyethoxylated castor oil formulation comprises about 50% ethanol andabout 50% polyethoxylated castor oil or about 85% ethanol and about 15%polyethoxylated castor oil.

The invention claimed is:
 1. A method for the treatment of cancer in apatient in need thereof, the method comprising administering to thepatient a therapeutically effective amount of a compound of FormulaS-(1):

wherein the compound of Formula S-(1) is administered in a compositionfurther comprising a pharmaceutically acceptable carrier.
 2. The methodof claim 1, wherein the pharmaceutically acceptable carrier comprises acomponent selected from the group consisting of a diluent, a binder, anadhesive, a disintegrant, a lubricant, an antiadherent, a glidant, asweetener, a flavorant, a colorant, a coating, a wetting agent, adispersant, a flocculation agent, a thickener, a buffer, an emulsifier,an amphiphilic, an osmotic agent, a coloring agent, an anti-oxidant, aflavor, a fragrance, and a preservative.
 3. The method of claim 1,wherein the pharmaceutically acceptable carrier comprises a componentselected from the group consisting of ethanol, polyethoxylated castoroil, vitamin E TPGS, oleic acid, polysorbate 80, polyethylene glycol,propylene glycol, and methylcellulose.
 4. The method of claim 1, whereinthe composition is administered orally.
 5. The method of claim 1,wherein the composition is administered intravenously.
 6. The method ofclaim 1, wherein the composition is administered parenterally.
 7. Themethod of claim 1, wherein the compound is administered to the patientat a dose of 240 mg/m² or less, 185 mg/m² or less, or 160 mg/m² or less.8. The method of claim 1, wherein the compound is administered to thepatient at a dose of 160 mg/m² or more, or 185 mg/m² or more.
 9. Themethod of claim 1, wherein the compound is administered to the patientat a dose ranging from 5 to 600 mg/m², from 25 to 400 mg/m², from 7 to555 mg/m², from 7 to 240 mg/m², or from 7 to 185 mg/m².
 10. The methodof claim 1, wherein the compound is administered to the patient at adose selected from the group consisting of about 7 mg/m², about 14mg/m², about 28 mg/m², about 56 mg/m², about 84 mg/m², about 126 mg/m²,about 127.5 mg/m², about 160 mg/m², about 185 mg/m², about 200 mg/m²,and about 220 mg/m².
 11. The method of claim 1, wherein the compound isadministered to the patient at a dose of about 160 mg/m².
 12. The methodof claim 1, wherein the compound is administered to the patient at adose of about 185 mg/m².
 13. The method of claim 1, wherein the compoundis administered to the patient at a dose of about 200 mg/m².
 14. Themethod of claim 1, wherein the compound is administered to the patientat a dose of about 220 mg/m².
 15. A treatment cycle comprisingadministration of the compound of Formula S-(1) according to the methodof claim 1, the cycle comprising at least one compound administration.16. The treatment cycle of claim 15, wherein the cycle further comprisesan additional compound administration.
 17. The treatment cycle of claim16, wherein the time period between administrations is about one week.18. The treatment cycle of claim 16, wherein the time period betweenadministrations is about two weeks.
 19. The treatment cycle of claim 16,wherein the time period between administrations is about three weeks.20. The method of claim 1, wherein the cancer is selected from the groupconsisting of brain cancer, hepatocellular cancer, sarcoma, leukemia,lymphoma, bone marrow dyscrasias, neuroblastoma, glioblastoma, cervicalcancer, colorectal cancer, pancreatic cancer, renal cancer, thyroidcancer, lung cancer, small-cell lung cancer, non-small cell lung cancer,gastric cancer, breast cancer, ovarian cancer, prostate cancer, head andneck cancer, and melanoma.
 21. The method of claim 1, wherein the canceris brain cancer.
 22. The method of claim 1, wherein the cancer isselected from the group consisting of astrocytoma, craniopharyngioma,glioma, ependymoma, neuroglioma, oligodendroglioma, glioblastomamultiforme, meningioma, medulloblastoma, and a primitive neuroectoderma.23. The method of claim 1, wherein the cancer is neuroblastoma.
 24. Themethod of claim 1, wherein the cancer is colorectal cancer.
 25. Themethod of claim 1, wherein the cancer is pancreatic cancer.